Hybrid polypeptides with selectable properties

ABSTRACT

The present invention relates generally to novel, selectable hybrid polypeptides useful as agents for the treatment and prevention of metabolic diseases and disorders which can be alleviated by control plasma glucose levels, insulin levels, and/or insulin secretion, such as diabetes and diabetes-related conditions. Such conditions and disorders include, but are not limited to, hypertension, dyslipidemia, cardiovascular disease, eating disorders, insulin-resistance, obesity, and diabetes mellitus of any kind, including type 1, type 2, and gestational diabetes.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/323,608, filed Dec. 12, 2011, which is a continuation of U.S.application Ser. No. 11/206,903 filed Aug. 17, 2005, entitled “HybridPolypeptides With Selectable Properties”, which is a continuation-inpart of U.S. application Ser. No. 11/201,664 filed Aug. 11, 2005, nowabandoned, which is a continuation-in-part of U.S. application Ser. No.11/055,093, filed Feb. 11, 2005, now abandoned, which claims priority tocommonly-owned U.S. Provisional Application No. 60/543,407, filed Feb.11, 2004, the disclosures of which are hereby incorporated by referencein their entirety, and to each of which priority is claimed.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. The ASCII copy, created on Feb. 25, 2014, isnamed “92494-901893_ST25.txt” and is 175,233 bytes in size.

FIELD OF THE INVENTION

The present invention relates to peptide chemistry, and moreparticularly to hybrid polypeptides with selectable properties.

BACKGROUND OF THE INVENTION

Central to many metabolic diseases and disorders is the regulation ofinsulin levels and blood glucose levels. Insulin secretion is modulatedin part by secretagogue hormones, termed as incretins, which areproduced by enteroendocrine cells. The incretin hormone, glucagon-likepeptide-1 (“GLP-1”) is a peptide hormone secreted by intestinal cellsthat has been shown in multiple studies to produce an enhancing effecton insulin secretion. GLP-1 is processed from proglucagon in the gut andenhances nutrient-induced insulin release (Krcymann B., et al., Lancet,2:1300-1303 (1987)). Various truncated forms of GLP-1, are known tostimulate insulin secretion (insulinotropic action) and cAMP formation(see, e.g., Mojsov, S., Int. J. Pep. Pro. Res., 40:333-343 (1992)). Arelationship between various in vitro laboratory experiments andmammalian, especially human, insulinotropic responses to exogenousadministration of GLP-1, GLP-1(7-36) amide (SEQ ID NO:61), andGLP-1(7-37) acid (SEQ ID NO:204) has been established (see, e.g., Nauck,M. A., et al., Diabetologia, 36:741-744 (1993); Gutniak, M., et al., NewEng. J. of Med., 326(20):1316-1322 (1992); Nauck, M. A., et al., J.Clin. Invest., 91:301-307 (1993); and Thorens, B., et al., Diabetes,42:1219-1225 (1993)).

GLP-1(7-36) amide (SEQ ID NO:61) exerts a pronounced antidiabetogeniceffect in insulin-dependent diabetics by stimulating insulin sensitivityand by enhancing glucose-induced insulin release at physiologicalconcentrations (Gutniak M., et al., New Eng. J. Med., 326:1316-1322(1992)). When administered to non-insulin dependent diabetics,GLP-1(7-36) amide (SEQ ID NO:61) stimulates insulin release, lowersglucagon secretion, inhibits gastric emptying and enhances glucoseutilization (Nauck, 1993; Gutniak, 1992; Nauck, 1993). However, the useof GLP-1 type molecules for prolonged therapy of diabetes has beencomplicated because the serum half-life of such peptides is quite short.

More particularly, GLP-1 is a 30-amino acid peptide derived fromproglucagon, a 160-amino acid prohormone. Actions of differentprohormone convertases in the pancreas and intestine result in theproduction of glucagon and other ill-defined peptides, whereas cleavageof proglucagon results in the production of GLP-1 and GLP-2 as well astwo other peptides. The amino acid sequence of GLP-1 is 100% homologousin all mammals studied so far, implying a critical physiological role.GLP-1 (7-37) acid is C-terminally truncated and amidated to form GLP-1(7-36) NH₂ (SEQ ID NO:61). The biological effects and metabolic turnoverof the free acid GLP-1 (7-37) OH (SEQ ID NO:204), and the amide, GLP-1(7-36) NH₂ (SEQ ID NO:61), are indistinguishable. By convention, thenumbering of the amino acids is based on the processed GLP-1 (1-37) OH(SEQ ID NO:59) from proglucagon. The biologically active GLP-1 is theresult of further processing: GLP-1 (7-36) NH₂ (SEQ ID NO:61). Thus thefirst amino acid of GLP-1 (7-37) OH (SEQ ID NO:204) or GLP-1 (7-36)NH₂(SEQ ID NO:61) is ⁷His.

In the gastrointestinal tract, GLP-1 is produced by L-cells ofintestinal, colonic and rectal mucosa, in response to stimulation byintraluminal glucose. The plasma half-life of active GLP-1 is <5minutes, and its metabolic clearance rate is around 12-13 minutes(Holst, Gastroenterology 107(6):1848-55 (1994)). The major proteaseinvolved in the metabolism of GLP-1 is dipeptidyl peptidase (DPP) IV(CD26) which cleaves the N-terminal His-Ala dipeptide, thus producingmetabolites, GLP-1 (9-37) OH (SEQ ID NO:205) or GLP-1 (9-36) NH₂ (SEQ IDNO:206) which are variously described as inactive, weak agonist orantagonists of GLP-1 receptor. The GLP-1 receptor (GLP-1R) is a Gprotein coupled receptor of 463 amino acid and is localized inpancreatic beta cells, in the lungs, and to a lesser extent in thebrain, adipose tissue and kidneys. The stimulation of GLP-1R by GLP-1(7-37) OH (SEQ ID NO:204) or GLP-1 (7-36)NH₂ (SEQ ID NO:61) results inadenylate cyclase activation, cAMP synthesis, membrane depolarization,rise in intracellular calcium and increase in glucose-induced insulinsecretion (Holz et al., J. Biol. Chem. 270(30):17749-57 (1995)).

GLP-1 is a potent insulin secretagogue that is secreted from theintestinal mucosa in response to food intake. The profound incretineffect of GLP-1 is underscored by the fact that GLP-1R knockout mice areglucose-intolerant. The incretin response of i.v. infused GLP-1 ispreserved in diabetic subjects, though the incretin response to oralglucose in these patients is compromised. GLP-1 administration byinfusion or sc injections controls fasting glucose levels in diabeticpatients, and maintains the glucose threshold for insulin secretion(Gutniak et al., N. Engl. J. Med. 326:1316-22 (1992); Nauck et al.,Diabet. Med. 13:(9 Suppl 5):539-S43 (1996); Nauck et al., J. Clin.Endocrinol. Metab. 76:912-917 (1993)). GLP-1 has shown tremendouspotential as a therapeutic agent capable of augmenting insulin secretionin a physiological manner, while avoiding hypoglycemia associated withsulfonylurea drugs.

Other important effects of GLP-1 on glucose homeostasis are suppressionof glucagon secretion and inhibition of gastric motility. GLP-1inhibitory actions on pancreatic alpha cell secretion of glucagon leadsto decreases in hepatic glucose production via reduction ingluconeogenesis and glycogenolysis. This antiglucagon effect of GLP-1 ispreserved in diabetic patients.

The so-called ileal brake effect of GLP-1, in which gastric motility andgastric secretion are inhibited, is effected via vagal efferentreceptors or by direct action on intestinal smooth muscle. Reduction ofgastric acid secretion by GLP-1 contributes to a lag phase in nutrientavailability, thus obviating the need for rapid insulin response. Insummary, the gastrointestinal effects of GLP-1 contribute significantlyto delayed glucose and fatty acid absorption and modulate insulinsecretion and glucose homeostasis.

GLP-1 has also been shown to induce beta cell specific genes, such asGLUT-1 transporter, insulin (via the interaction of PDX-1 with insulingene promoter), and hexokinase-1. Thus GLP-1 could potentially reverseglucose intolerance normally associated with aging, as demonstrated byrodent experiments. In addition, GLP-1 may contribute to beta cellneogenesis and increase beta cell mass, in addition to restoring betacell function during states of beta cell insufficiency.

Central effects of GLP-1 include increases in satiety coupled withdecreases in food intake, effected via the action of hypothalamicGLP-1R. A 48 hour continuous SC infusion of GLP-1 in type II diabeticsubjects, decreased hunger and food intake and increased satiety. Theseanorectic effects were absent in GLP-1R knock out mice.

Exendins are another family of peptides implicated in insulin secretion.Exendins are found in the saliva of the Gila-monster, a lizardendogenous to Arizona, and the Mexican Beaded Lizard. Exendin-3 ispresent in the saliva of Heloderma horridum, and exendin-4 is present inthe saliva of Heloderma suspectum (Eng, J., et al., J. Biol. Chem.,265:20259-62, 1990; Eng., J., et al., J. Biol. Chem., 267:7402-05(1992)). The exendins have some sequence similarity to several membersof the glucagon-like peptide family, with the highest identity, 53%,being to GLP-1 (Goke, et al., J. Biol. Chem., 268:19650-55 (1993)).

Exendin-4 binds the GLP-1 receptors on insulin-secreting TC1 cells, atdispersed acinar cells from guinea pig pancreas, and at parietal cellsfrom stomach; the peptide also stimulates somatostatin release andinhibits gastrin release in isolated stomachs (Goke, et al., J. Biol.Chem., 268:19650-55 (1993); Schepp, et al., Eur. J. Pharmacol.,69:183-91 (1994); Eissele, et al., Life Sci., 55:629-34 (1994)).Exendin-3 and exendin-4 were found to bind the GLP-1 receptors on, tostimulating cAMP production in, and amylase release from, pancreaticacinar cells (Malhotra, R., et al., Relulatory Peptides, 41:149-56(1992); Raufman, et al., J. Biol. Chem., 267:21432-37 (1992); Singh, etal., Regul. Pept., 53:47-59 (1994)). The use of the insulinotropicactivities of exendin-3 and exendin-4 for the treatment of diabetesmellitus and the prevention of hyperglycemia has been proposed (Eng,U.S. Pat. No. 5,424,286).

Truncated exendin peptides such as exendin[9-39], a carboxyamidatedmolecule, and fragments 3-39 through 9-39 have been reported to bepotent and selective antagonists of GLP-1 (Goke, et al., J. Biol. Chem.,268:19650-55 (1993); Raufman, J. P., et al., J. Biol. Chem.,266:2897-902 (1991); Schepp, W., et al., Eur. J. Pharm., 269:183-91(1994); Montrose-Rafizadeh, et al., Diabetes, 45(Suppl. 2):152A (1996)).Exendin[9-39] (SEQ ID NO:207) blocks endogenous GLP-1 in vivo, resultingin reduced insulin secretion (Wang, et al., J. Clin. Invest., 95:417-21(1995); D'Alessio, et al., J. Clin. Invest., 97:133-38 (1996)). Thereceptor apparently responsible for the insulinotropic effect of GLP-1has been cloned from rat pancreatic islet cells (Thorens, B., Proc.Natl. Acad. Sci. USA 89:8641-8645 (1992)). Exendins and exendin[9-39]bind to the cloned GLP-1 receptor (rat pancreatic-cell GLP-1 receptor:Fehmann H C, et al., Peptides, 15 (3): 453-6 (1994); human GLP-1receptor: Thorens B, et al., Diabetes, 42 (11): 1678-82 (1993)). Incells transfected with the cloned GLP-1 receptor, exendin-4 is anagonist, i.e., it increases cAMP, while exendin[9-39] (SEQ ID NO:207) isan antagonist, i.e., it blocks the stimulatory actions of exendin-4 andGLP-1. Id.

More particularly, exendin-4 is a 39 amino acid C-terminal amidatedpeptide found in the saliva of the Gila Monster (Heloderma suspectum),with a 53% amino acid sequence identity to the GLP-1 peptide sequence.See, e.g., Eng, J., et al. “Isolation and Characterization of Exendin-4,and Exendin-3 Analogue from Heloderma suspectum Venom,” J. Bio. Chem.,267:11, p. 7402-7405 (1992), Young, A. A., et al., “Glucose-Lowering andInsulin-Sensitizing Actions of Exendin-4,” Diabetes, Vol. 48, p.1026-1034, May, 1999. In terms of its activity, exendin-4 is a highlyspecific agonist for the GLP-1 receptor, and, like GLP-1, is able tostimulate insulin secretion. Therefore, like GLP-1, exendin-4 isregarded as an insulinotropic peptide.

However, unlike GLP-1, exendin-4 has a relatively long half-life inhumans, because of its resistance to the dipeptidyl peptidase IV whichrapidly degrades the GLP-1 sequence in vivo. Furthermore, it has beenshown that, as compared to GLP-1, exendin-4 has a stronger capability tostimulate insulin secretion, and that a lower concentration of exendin-4may be used to obtain such stimulating activity. See, e.g., U.S. Pat.No. 5,424,286, herein incorporated by reference. Therefore exendin-4peptides or derivatives thereof (for examples of such derivatives, see,e.g., U.S. Pat. No. 6,528,486, herein incorporated by reference, and itscorresponding international application WO 01/04156) have a greaterpotential utility for the treatment of conditions involving thedysregulation of insulin levels (e.g., conditions such as diabetes) thaneither insulin or GLP-1.

Another family of peptide hormones implicated in metabolic diseases anddisorders is the amylin family of peptide hormones, including amylin,calcitonin, calcitonin gene related peptide, adrenomedullin, andintermedin (also known as “AFP-6”). Amylin is a 37-amino acid peptidehormone. It was isolated, purified and chemically characterized as themajor component of amyloid deposits in the islets of pancreases of humanType 2 diabetics (Cooper et al., Proc. Natl. Acad. Sci., USA,84:8628-8632 (1987)). The amylin molecule has two post-translationalmodifications: the C-terminus is amidated, and the cysteines inpositions 2 and 7 are cross-linked to form an N-terminal loop. Thesequence of the open reading frame of the human amylin gene shows thepresence of the Lys-Arg dibasic amino acid proteolytic cleavage signal,prior to the N-terminal codon for Lys, and the Gly prior to the Lys-Argproteolytic signal at the CLAIMS-terminal position, a typical sequencefor amidation by protein amidating enzyme, PAM (Cooper et al., Biochem.Biophys. Acta, 1014:247-258 (1989)).

Amylin is believed to regulate gastric emptying, and suppress glucagonsecretion and food intake, thus regulating the rate of glucoseappearance in the circulation. It appears to complement the actions ofinsulin, which regulates the rate of glucose disappearance from thecirculation and its uptake by peripheral tissues. These actions aresupported by experimental findings in rodents and humans, which indicatethat amylin complements the effects of insulin in postprandial glucosecontrol by at least three independent mechanisms, all of which affectthe rate of glucose appearance. First, amylin suppresses postprandialglucagon secretion. Compared to healthy adults, patients with type 1diabetes have no circulating amylin and patients with type 2 diabeteshave diminished postprandial amylin concentrations. Furthermore,infusion of an amylin specific monoclonal antibody, which boundcirculating amylin, again resulted in greatly elevated glucagonconcentrations relative to controls. Both of these results point to aphysiological role of endogenous amylin in the regulation ofpostprandial glucagon secretion. Second, amylin slows gastrointestinalmotility and gastric emptying. Finally, intrahypothalamic injections ofrat amylin were shown to reduce feeding in rats and alterneurotransmitter metabolism in the hypothalamus. In certain studies,food intake was significantly reduced for up to eight hours followingthe intrahypothalamic injection of rat amylin and rat CGRP. In humantrials, an amylin analog, pramlintide, has been shown to reduce weightor weight gain. Amylin may be beneficial in treating metabolicconditions such as diabetes and obesity. Amylin may also be used totreat pain, bone disorders, gastritis, to modulate lipids, in particulartriglycerides, or to affect body composition such as the preferentialloss of fat and sparing of lean tissue.

The hormone calcitonin (CT) was named for its secretion in response toinduced hypercalcemia and its rapid hypocalcemic effect. It is producedin and secreted from neuroendocrine cells in the thyroid that have sincebeen termed C cells. The best-studied action of CT(1-32) (SEQ ID NO:48)is its effect on the osteoclast. In vitro effects of CT include therapid loss of ruffled borders and decreased release of lysosomalenzymes. Ultimately, the inhibition of osteoclast functions by CTresults in a decrease in bone resorption. However, neither a chronicreduction of serum CT in the case of thyroidectomy nor the increasedserum CT found in medullary thyroid cancer appears to be associated withchanges in serum calcium or bone mass. It is thus most likely that amajor function of CT(1-32) (SEQ ID NO:48) is to combat acutehypercalcemia in emergency situations and/or protect the skeleton duringperiods of “calcium stress” such as growth, pregnancy, and lactation.(Reviewed in Becker, JCEM, 89(4): 1512-1525 (2004) and Sexton, CurrentMedicinal Chemistry 6: 1067-1093 (1999)). Consistent with this is recentdata from the calcitonin gene knockout mouse, which removes both thecalcitonin and the CGRP-I peptides, that revealed that the mouse hadnormal levels of basal calcium-related values, but an increased calcemicresponse (Kurihara H, et al., Hypertens Res. 2003 February; 26Suppl:S105-8).

CT has an effect on plasma calcium levels and inhibits osteoclastfunction and is widely used for the treatment of osteoporosis.Therapeutically, salmon CT (sCT) appears to increase bone density anddecrease fracture rates with minimal adverse effects. CT has also beensuccessfully used over the past 25 years as a therapy for Paget'sdisease of bone, which is a chronic skeletal disorder that may result inenlarged or deformed bones in one or more regions of the skeleton. CT isalso widely used for its analgesic effect on bone pain experiencedduring osteoporosis, although the mechanism for this effect is notclearly understood.

Calcitonin gene related peptide (CGRP) is a neuropeptide whose receptorsare widely distributed in the body, including the nervous system and thecardiovascular system. This peptide seems to modulate sensoryneurotransmission and is one of the most potent endogenous vasodilatorypeptide discovered to date. Reported biological effects for CGRPinclude: modulation of substance P in inflammation, nicotinic receptoractivity at the neuromuscular junction, stimulation of pancreatic enzymesecretion, a reduction of gastric acid secretion, peripheralvasodilation, cardiac acceleration, neuro-modulation, regulation ofcalcium metabolism, osteogenic stimulation, insulin secretion, anincrease in body temperature and a decrease in food intake.(Wimalawansa, Amylin, calcitonin gene-related peptide, calcitonin andADM: a peptide superfamily. Crit Rev Neurobiol. 1997; 11(2-3):167-239).An important role of CGRP is to control blood flow to various organs byits potent vasodilatory actions, as evidenced by a decrease of meanarterial pressure following intravenous administration of α-CGRP. Thevasodilatory actions are also supported by recent analysis of homozygousknockout CGRP mice, which demonstrated elevated peripheral vascularresistance and high blood pressure caused by increased peripheralsympathetic activity (Kurihara H, et al., Targeted disruption of ADM andαCGRP genes reveals their distinct biological roles. Hypertens Res. 2003February; 26 Suppl:S105-8). Thus, CGRP appears to elicit vasodilatoryeffects, hypotensive effects and an increase in heart rate among otheractions.

Prolonged infusion of CGRP into patients with congestive cardiac failurehas shown a sustained beneficial effect on hemodynamic functions withoutadverse effects, suggesting a use in heart failure. Other indications ofCGRP use include renal failure, acute and chronic coronary arteryischemia, treatment of cardiac arrhythmia, other peripheral vasculardisease such as Raynaud's phenomenon, subarachnoid hemorrhage,hypertension, and pulmonary hypertension. Preeclamptic toxemia ofpregnancy and preterm labor are also potentially treatable.(Wimalawansa, 1997). Recent therapeutic uses include the use of CGRPantagonists for the treatment of migraine headaches.

Adrenomedullin (ADM) is almost ubiquitously expressed with many moretissues containing the peptide than not. A published review of ADM,(Hinson, J. P. et al., Endocrine Reviews (2000) 21(2): 138-167) detailsits effects on the cardiovascular system, cellular growth, the centralnervous system and the endocrine system, with a range of biologicalactions including vasodilation, cell growth, regulation of hormonesecretion, and natriuresis. Studies in rat, cat, sheep, and man confirmthat intravenous infusion of ADM results in potent and sustainedhypotension, and is comparable to that of CGRP. However, the hypotensiveeffect of ADM on mean arterial pressure in the anesthetized rat is notinhibited by the CGRP antagonist CGRP₈₋₃₇ suggesting that this effect isnot mediated via CGRP receptors. Acute or chronic administration ofhuman ADM in rats, anesthetized, conscious or hypertensive, results in asignificant decrease in total peripheral resistance accompanied by afall in blood pressure, with a concomitant rise in heart rate, cardiacoutput and stroke volume.

ADM has also been proposed as an important factor in embryogenesis anddifferentiation and as an apoptosis survival factor for rat endothelialcells. This is supported by recent mouse ADM knockout studies, in whichmice homozygous for loss of the ADM gene demonstrated defective vascularformation during embryogenesis and thus died mid-gestation. It wasreported that ADM+/−heterozygous mice had high blood pressure along withsusceptibility to tissue injury (Kurihara H, et al., Hypertens Res. 2003February; 26 Suppl:S105-8).

ADM affects such endocrine organs as the pituitary, the adrenal gland,reproductive organs and the pancreas. The peptide appears to have a rolein inhibiting ACTH release from the pituitary. In the adrenal gland, itappears to affect the secretory activity of the adrenal cortex in bothrat and human and it increases adrenal blood flow, acting as avasodilator in the adrenal vascular bed in intact rats. ADM has beenshown to be present throughout the female reproductive tract and plasmalevels are elevated in normal pregnancy. Studies in a rat model ofpreeclampsia show that ADM can reverse hypertension and decrease pupmortality when given to rats during late gestation. Because it did nothave a similar effect in animals in early gestation or non-pregnant ratsin the preeclampsia model, this suggests that ADM may play an importantregulatory role in the utero-placental cardiovascular system. In thepancreas, ADM most likely plays an inhibitory role since it attenuatedand delayed insulin response to an oral glucose challenge, resulting ininitial elevated glucose levels. ADM can also affect renal function. Abolus administered peripherally can significantly lower mean arterialpressure and raise renal blood flow, glomerular filtration rate andurine flow. In some cases, there is also an increase in Na+ excretion.

ADM also has other peripheral effects on bone and on the lung. For bone,studies have supported a role beyond the cardiovascular system and fluidhomeostasis and have demonstrated that ADM acts on fetal and adultrodent osteoblasts to increase cell growth comparable to those of knownosteoblast growth factors such as transforming growth factor-β. This isimportant clinically as one of the major challenges in osteoporosisresearch is to develop a therapy that increases bone mass viaosteoblastic stimulation. In the lung, ADM not only causes pulmonaryvasodilation, but also inhibits bronchoconstriction induced by histamineor acetylcholine. Recent studies using aerosolized ADM to treatpulmonary hypertension in a rat model indicate that inhalation treatmentof this condition is effective, as evidenced by the fact that meanpulmonary arterial pressure and total pulmonary resistance were markedlylower in rats treated with ADM than in those given saline. This resultwas achieved without an alteration in systemic arterial pressure orheart rate (Nagaya N et al., Am J Physiol Heart Circ Physiol. 2003;285:H2125-31).

In healthy volunteers, i.v. infusion of ADM has been shown to reducearterial pressure and to stimulate heart rate, cardiac output, plasmalevels of cAMP, prolactin, norepinephrine and rennin. In these patients,there was little or no increase in urine volume or sodium excretionobserved. In patients with heart failure or chronic renal failure, i.v.ADM had similar effects to those seen in normal subjects, and alsoinduced diuresis and natriuresis, depending on the dose administered(Nicholls, M G et al. Peptides. 2001; 22:1745-1752) Experimental ADMtreatment has also been shown to be beneficial in arterial and pulmonaryhypertension, septic shock and ischemia/reperfusion injury (BeltowskiJ., Pol J Pharmacol. 2004; 56:5-27). Other indications for ADM treatmentinclude: peripheral vascular disease, subarachnoid hemorrhage,hypertension, preeclamptic toxemia of pregnancy and preterm labor, andosteoporosis.

Expression of AFP-6 (i.e., intermedin) is primarily in the pituitary andgastrointestinal tract. A specific receptor for AFP-6 has not beenreported; however, binding studies indicate that AFP-6 binds to all theknown receptors of the Amylin Family. AFP-6 has been shown to increasecAMP production in SK-N-MC and L6 cells expressing endogenous CGRPreceptors and competes with labeled CGRP for binding to its receptors inthese cells. In published in vivo studies, AFP-6 administration led toblood pressure reduction in both normal and spontaneously hypertensiverats, most likely via interactions with the CRLR/RAMP receptors. In vivoadministration in mice led to a suppression of gastric emptying and foodintake. (Roh et al. J Biol Chem. 2004 Feb. 20; 279(8):7264-74.)

It has been reported that the biological actions of amylin familypeptide hormones are generally mediated via binding to two closelyrelated type II G protein-coupled receptors (GPCRs), the calcitoninreceptor (CTR) and the calcitonin receptor like receptor (CRLR). Cloningand functional studies have shown that CGRP, ADM, and amylin interactwith different combinations of CTR or the CRLR and the receptor activitymodifying protein (RAMP). Many cells express multiple RAMPs. It isbelieved that co-expression of RAMPs and either the CTR or CRLR isrequired to generate functional receptors for calcitonin, CGRP, ADM, andamylin. The RAMP family comprises three members (RAMP1, -2, and -3),which share less then 30% sequence identity, but have a commontopological organization. Co-expression of CRLR and RAMP1 leads to theformation of a receptor for CGRP. Co-expression of CRLR and RAMP2 leadsto the formation of a receptor for ADM. Co-expression of CRLR and RAMP3leads to the formation of a receptor for ADM and CGRP. Co-expression ofhCTR2 and RAMP1 leads to the formation of a receptor for amylin andCGRP. Co-expression of hCTR2 and RAMP3 leads to the formation of areceptor for amylin.

Yet another peptide hormone family implicated in metabolic diseases anddisorders is the leptin family. The mature form of circulating leptin isa 146-amino acid protein that is normally excluded from the CNS by theblood-brain barrier (BBB) and the blood-CSF barrier. See, e.g., Weigleet al., 1995. J Clin Invest 96: 2065-2070. Leptin is the afferent signalin a negative feedback loop regulating food intake and body weight. Theleptin receptor is a member of the cytokine receptor family. Leptin'sanorexigenic effect is dependent on binding to homodimer of the Ob-Rbisoform of this receptor which encodes a long intra-cytoplasmic domainthat includes several motifs for protein-protein interaction. Ob-Rb ishighly expressed in the hypothalamus suggesting that this brain regionis an important site of leptin action. Mutation of the mouse ob gene hasbeen demonstrated to result in a syndrome that exhibits-pathophysiologythat includes: obesity, increased body fat deposition, hyperglycemia,hyperinsulinemia, hypothermia, and impaired thyroid and reproductivefunctions in both male and female homozygous ob/ob obese mice (see e.g.,Ingalis, et al., 1950. J Hered 41: 317-318. Therapeutic uses for leptinor leptin receptor include (i) diabetes (see, e.g., PCT PatentApplications WO 98/55139, WO 98/12224, and WO 97/02004); (ii)hematopoiesis (see, e.g., PCT Patent Applications WO 97/27286 and WO98/18486); (iii) infertility (see, e.g., PCT Patent Applications WO97/15322 and WO 98/36763); and (iv) tumor suppression (see, e.g., PCTPatent Applications WO 98/48831), each of which are incorporated hereinby reference in their entirety.

The leptin receptor (OB-R) gene has been cloned (GenBank Accession No.AF098792) and mapped to the db locus (see, e.g., Tartaglia, et al.,1995. Cell 83: 1263-1271). Several transcripts of the OB-R, resultingfrom alternative splicing, have also been identified. Defects in OB-Rproduce a syndrome in the mutant diabetic ob/ob mouse that isphenotypically identical to the ob/ob mouse (see, e.g., Ghilardi, etal., 1996. Proc. Natl. Acad. Sci. USA 93: 6231-6235). In contrast toob/ob mice, however, administration of recombinant leptin to C57BLKS/J-mob/ob mice does not result in reduced food intake and body weight (see,e.g., Roberts and Greengerg, 1996. Nutrition Rev. 54: 41-49).

Most leptin-related studies able to report weight loss activity fromadministration of recombinant leptin, leptin fragments and/or leptinreceptor variants have administered said constructs directly into theventricles of the brain. See e.g., Weigle, et al., 1995. J Clin Invest96: 2065-2070; Barash, et al., 1996. Endocrinology 137: 3144-3147.

Other studies have shown significant weight loss activity due toadministration of leptin peptides through intraperitoneally (i.p.)administration to test subjects. See, Grasso et al., 1997. Endocrinology138: 1413-1418. Further, leptin fragments, and most particularly an 18amino acid fragment comprising residues taken from full length humanleptin, have been reported to function in weight loss, but only upondirect administration through an implanted cannula to the lateral brainventricle of rats. See, e.g., PCT Patent Applications WO 97/46585, whichis incorporated herein by reference in its entirety.

Another peptide hormone implicated in metabolic diseases and disordersis cholecystokinin (CCK). CCK was reportedly identified in 1928 frompreparations of intestinal extracts by its ability to stimulategallbladder contraction. Other biological actions of CCK have since beenreported, including stimulation of pancreatic secretion, delayed gastricemptying, stimulation of intestinal motility and stimulation of insulinsecretion. See Lieverse et al., Ann. N.Y. Acad. Sci. 713: 268-272(1994). The actions of CCK, also reportedly include effects oncardiovascular function, respiratory function, neurotoxicity andseizures, cancer cell proliferation, analgesia, sleep, sexual andreproductive behaviors, memory, anxiety and dopamine-mediated behaviors.Crawley and Corwin, Peptides 15: 731-755 (1994). Other reported effectsof CCK include stimulation of pancreatic growth, stimulation ofgallbladder contraction, inhibition of gastric acid secretion,pancreatic polypeptide release and a contractile component ofperistalsis. Additional reported effects of CCK include vasodilation.Walsh, “Gastrointestinal Hormones,” In Physiology of theGastrointestinal Tract (3d ed. 1994; Raven Press, New York).

It has been reported that injections of combinations of glucagon, CCKand bombesin potentiated the inhibition of intake of condensed milk testmeals in nondeprived rats over the inhibitions observed with individualcompounds. Hinton et al., Brain Res. Bull. 17:615-619 (1986). It hasalso been reported that glucagon and CCK synergistically inhibit shamfeeding in rats. LeSauter and Geary, Am. J. Physiol. 253:R217-225(1987); Smith and Gibbs, Annals N.Y. Acad. Sci. 713:236-241 (1994). Ithas also been suggested that estradiol and CCK can have a synergisticeffect on satiety. Dulawa et al., Peptides 15:913-918 (1994); Smith andGibbs, supra. It has also been proposed that signals arising from thesmall intestine in response to nutrients therein may interactsynergistically with CCK to reduce food intake. Cox, Behav. Brain Res.38:35-44 (1990). Additionally, it has been reported that CCK inducessatiety in several species. For example, it has been reported thatfeeding depression was caused by CCK injected intraperitoneally in rats,intraarterially in pigs, intravenously in cats and pigs, into thecerebral ventricles in monkeys, rats, dogs and sheep, and intravenouslyin obese and non-obese humans. See Lieverse et al., supra. Studies fromseveral laboratories have reportedly confirmed the behavioralspecificity of low doses of CCK on inhibition in feeding, by comparingresponding for food to responding for nonfood reinforcers in bothmonkeys and rats and by showing that CCK elicits the sequence ofbehaviors normally observed after meal ingestion (i.e., the postprandialsatiety sequence). Additionally, comparison of behavior after CCK tobehavior after food ingestion, alone or in combination with CCK hasreportedly revealed behavioral similarities between CCK and foodingestion. Crawley and Corwin, supra. It has also been reported that CCKin physiological plasma concentrations inhibits food intake andincreases satiety in both lean and obese humans. See Lieverse et al.,supra.

CCK was characterized in 1966 as a 33-amino acid peptide. Crawley andCorwin, supra. Species-specific molecular variants of the amino acidsequence of CCK have been identified. The 33-amino acid sequence and atruncated peptide, its 8-amino acid C-terminal sequence (CCK-8) havebeen reportedly identified in pig, rat, chicken, chinchilla, dog andhumans. A 39-amino acid sequence was reportedly found in pig, dog andguinea pig. A 58-amino acid sequence was reported to have been found incat, dog and humans. Frog and turtle reportedly show 47-amino acidsequences homologous to both CCK and gastrin. Very fresh human intestinehas been reported to contain small amounts of an even larger molecule,termed CCK-83. In the rat, a principal intermediate form has beenreportedly identified, and is termed CCK-22. Walsh, “GastrointestinalHormones,” In Physiology of the Gastrointestinal Tract (3d ed. 1994;Raven Press, New York). A non-sulfated CCK-8 and a tetrapeptide (termedCCK-4 (CCK(30-33); SEQ ID NO:208) have been reported in rat brain. TheC-terminal pentapeptide (termed CCK-4 (CCK(29-33); SEQ ID NO:209)conserves the structural homology of CCK, and also homology with theneuropeptide, gastrin. The C-terminal sulfated octapeptide sequence,CCK-8, is reportedly relatively conserved across species. Cloning andsequence analysis of a cDNA encoding preprocholecystokinin from ratthyroid carcinoma, porcine brain, and porcine intestine reportedlyrevealed 345 nucleotides coding for a precursor to CCK, which is 115amino acids and contains all of the CCK sequences previously reported tohave been isolated. Crawley and Corwin, supra.

CCK is said to be distributed throughout the central nervous system andin endocrine cells and enteric nerves of the upper small intestine. CCKagonists include CCK itself (also referred to as CCK-33), CCK-8(CCK(26-33); SEQ ID NO:55), non-sulfated CCK-8, pentagastrin (CCK-5 orCCK(29-33); SEQ ID NO:209), and the tetrapeptide, CCK-4 (CCK(30-33); SEQID NO:208). At the pancreatic CCK receptor, CCK-8 reportedly displacedbinding with a 1000-5000 greater potency than unsulfated CCK-8 or CCK-4,and CCK-8 has been reported to be approximately 1000-fold more potentthan unsulfated CCK-8 or CCK-4 in stimulating pancreatic amylasesecretion. Crawley and Corwin, supra. In homogenates from the cerebralcortex, CCK receptor binding was said to be displaced by unsulfatedCCK-8 and by CCK-4 at concentrations that were equimolar, 10-fold or100-fold greater than sulfated CCK-8. Id.

Receptors for CCK have been reportedly identified in a variety oftissues, and two primary subtypes have been described: type A receptorsand type B receptors. Type A receptors have been reported in peripheraltissues including pancreas, gallbladder, pyloric sphincter and afferentvagal fibers, and in discrete areas of the brain. The type A receptorsubtype (CCK_(A)) has been reported to be selective for the sulfatedoctapeptide. The Type B receptor subtype (CCK_(B)) has been identifiedthroughout the brain and in the stomach, and reportedly does not requiresulfation or all eight amino acids. See Reidelberger, J. Nutr. 124 (8Suppl.) 1327S-1333S (1994); Crawley and Corwin, supra.

Yet another family of peptide hormones implicated in metabolic diseasesand disorders is the pancreatic polypeptide family (“PPF”). Pancreaticpolypeptide (“PP”) was discovered as a contaminant of insulin extractsand was named by its organ of origin rather than functional importance(Kimmel et al., Endocrinology 83: 1323-30 (1968)). PP is a 36-amino acidpeptide containing distinctive structural motifs. A related peptide wassubsequently discovered in extracts of intestine and named Peptide YY(“PYY”) because of the N- and C-terminal tyrosines (Tatemoto, Proc.Natl. Acad. Sci. USA 79: 2514-8 (1982)). A third related peptide waslater found in extracts of brain and named Neuropeptide Y (“NPY”)(Tatemoto, Proc. Natl. Acad. Sci. USA 79: 5485-9 (1982); Tatemoto etal., Nature 296: 659-60 (1982)).

These three related peptides have been reported to exert variousbiological effects. Effects of PP include inhibition of pancreaticsecretion and relaxation of the gallbladder. Centrally administered PPproduces modest increases in feeding that may be mediated by receptorslocalized to the hypothalamus and brainstem (reviewed in Gehlert, Proc.Soc. Exp. Biol. Med. 218: 7-22 (1998)).

Release of PYY occurs following a meal. An alternate molecular form ofPYY is PYY(3-36) (SEQ ID NO:58) (Eberlein et al., Peptides 10: 797-803(1989); Grandt et al., Regul. Pept. 51: 151-9 (1994)). This fragmentconstitutes approximately 40% of total PYY-like immunoreactivity inhuman and canine intestinal extracts and about 36% of total plasma PYYimmunoreactivity in a fasting state to slightly over 50% following ameal. It is apparently a dipeptidyl peptidase-IV (DPP4) cleavage productof PYY. PYY(3-36) (SEQ ID NO:58) is reportedly a selective ligand at theY2 and Y5 receptors, which appear pharmacologically unique in preferringN-terminally truncated (i.e., C-terminal fragments of) NPY analogs.Peripheral administration of PYY reportedly reduces gastric acidsecretion, gastric motility, exocrine pancreatic secretion (Yoshinaga etal., Am. J. Physiol. 263: G695-701 (1992); Guan et al., Endocrinology128: 911-6 (1991); Pappas et al., Gastroenterology 91: 1386-9 (1986)),gallbladder contraction and intestinal motility (Savage et al., Gut 28:166-70 (1987)). The effects of central injection of PYY on gastricemptying, gastric motility and gastric acid secretion, as seen afterdirect injection in or around the hindbrain/brainstem (Chen and Rogers,Am. J. Physiol. 269: R787-92 (1995); Chen et al., Regul. Pept. 61: 95-98(1996); Yang and Tache, Am. J. Physiol. 268: G943-8 (1995); Chen et al.,Neurogastroenterol. Motil. 9: 109-16 (1997)), may differ from thoseeffects observed after peripheral injection. For example, centrallyadministered PYY had some effects opposite to those described herein forperipherally injected PYY(3-36) (SEQ ID NO:58) in that gastric acidsecretion was stimulated, not inhibited. Gastric motility was suppressedonly in conjunction with TRH stimulation, but not when administeredalone, and was indeed stimulatory at higher doses through presumedinteraction with PP receptors. PYY has been shown to stimulate food andwater intake after central administration (Morley et al., Brain Res.341: 200-3 (1985); Corp et al., Am. J. Physiol. 259: R317-23 (1990)).

Metabolic diseases and disorders take on many forms, including obesity,diabetes, dyslipidemia, insulin resistance, cellular apoptosis, etc.Obesity and its associated disorders are common and very serious publichealth problems in the United States and throughout the world. Upperbody obesity is the strongest risk factor known for type 2 diabetesmellitus, and is a strong risk factor for cardiovascular disease.Obesity is a recognized risk factor for hypertension, atherosclerosis,congestive heart failure, stroke, gallbladder disease, osteoarthritis,sleep apnea, reproductive disorders such as polycystic ovarian syndrome,cancers of the breast, prostate, and colon, and increased incidence ofcomplications of general anesthesia (see, e.g., Kopelman, Nature 404:635-43 (2000)). It reduces life-span and carries a serious risk ofco-morbidities above, as well disorders such as infections, varicoseveins, acanthosis nigricans, eczema, exercise intolerance, insulinresistance, hypertension hypercholesterolemia, cholelithiasis,orthopedic injury, and thromboembolic disease (Rissanen et al., Br. Med.J. 301: 835-7 (1990)). Obesity is also a risk factor for the group ofconditions called insulin resistance syndrome, or “Syndrome X.” Recentestimate for the medical cost of obesity and associated disorders is$150 billion worldwide. The pathogenesis of obesity is believed to bemultifactorial but the basic problem is that in obese subjects nutrientavailability and energy expenditure do not come into balance until thereis excess adipose tissue. Obesity is currently a poorly treatable,chronic, essentially intractable metabolic disorder. A therapeutic druguseful in weight reduction of obese persons could have a profoundbeneficial effect on their health.

Diabetes is a disorder of carbohydrate metabolism characterized byhyperglycemia and glucosuria resulting from insufficient production orutilization of insulin. Diabetes severely affects the quality of life oflarge parts of the populations in developed countries. Insufficientproduction of insulin is characterized as type 1 diabetes andinsufficient utilization of insulin is type 2 diabetes. However, it isnow widely recognized that there are many distinct diabetes relateddiseases which have their onset long before patients are diagnosed ashaving overt diabetes. Also, the effects from the suboptimal control ofglucose metabolism in diabetes gives rise to a wide spectrum of relatedlipid and cardiovascular disorders.

Dyslipidemia, or abnormal levels of lipoproteins in blood plasma, is afrequent occurrence among diabetics. Dyslipidemia is typicallycharacterized by elevated plasma triglycerides, low HDL (High DensityLipoprotein) cholesterol, normal to elevated levels of LDL (Low DensityLipoprotein) cholesterol and increased levels of small dense, LDL (LowDensity Lipoprotein) particles in the blood. Dyslipidemia is one of themain contributors to the increased incidence of coronary events anddeaths among diabetic subjects. Epidemiological studies have confirmedthis by showing a several-fold increase in coronary deaths amongdiabetic subjects when compared with non-diabetic subjects. Severallipoprotein abnormalities have been described among diabetic subjects.

Insulin resistance is the diminished ability of insulin to exert itsbiologically action across a broad range of concentrations. In insulinresistance, the body secretes abnormally high amounts of insulin tocompensate for this defect and a state of impaired glucose tolerancedevelops. Failing to compensate for the defective insulin action, theplasma glucose concentration inevitable rises, resulting in the clinicalstate of diabetes. It is being recognized that insulin resistance andrelative hyperinsulinemia have a contributory role in obesity,hypertension, atherosclerosis and type 2 diabetes. The association ofinsulin resistance with obesity, hypertension and angina has beendescribed as a syndrome, Syndrome X, having insulin resistance as thecommon pathogenic link.

Apoptosis is an active process of cellular self-destruction that isregulated by extrinsic and intrinsic signals occurring during normaldevelopment. It is well documented that apoptosis plays a key role inregulation of pancreatic endocrine beta cells. There is increasingevidence that in adult mammals the beta-cell mass is subject to dynamicchanges to adapt insulin production for maintaining euglycemia inparticular conditions, such as pregnancy and obesity. The control ofbeta cell mass depends on a subtle balance between cell proliferation,growth and programmed cell death (apoptosis). A disturbance of thisbalance may lead to impairment of glucose homeostasis. For example, itis noteworthy that glucose intolerance develops with aging when betacell replication rates are reduced and human autopsy studies repeatedlyshowed a 40-60% reduction of beta cell mass in patients withnon-insulin-dependent-diabetes mellitus compared with nondiabeticsubjects. It is generally agreed that insulin resistance is aninvariable accompaniment of obesity but that normoglycemia is maintainedby compensatory hyperinsulinemia until the beta cells become unable tomeet the increased demand for insulin, at which point type 2 diabetesbegins.

Attempts to treat the multiple abnormalities associated with diabeteshave prompted for the administration of several anti-diabeticmedicaments in order to address these abnormalities in the differentpatients. Examples of anti-diabetic medicaments are proteins such asinsulin and insulin analogues, and small molecules such as insulinsensitizers, insulin secretagogues and appetite regulating compounds.

There remains a need to develop polypeptides useful in the abovedescribed metabolic diseases, conditions, and disorders. Accordingly, itis an object of the present invention to provide hybrid polypeptides andmethods for producing and using them. The compounds of the inventionfind use in the metabolic diseases, conditions, and disorders describedabove and herein.

All documents referred to herein are incorporated by reference into thepresent application as though fully set forth herein.

BRIEF SUMMARY OF THE INVENTION

The present invention relates generally to novel, selectable hybridpolypeptides useful as agents for the treatment and prevention ofmetabolic diseases and disorders which can be alleviated by control ofplasma glucose levels, insulin levels, and/or insulin secretion, such asdiabetes and diabetes-related conditions. Such conditions and disordersinclude, but are not limited to, hypertension, dyslipidemia,cardiovascular disease, eating disorders, insulin-resistance, obesity,and diabetes mellitus of any kind, including type 1, type 2, andgestational diabetes.

In one aspect of the invention, hybrid polypeptides exhibiting at leastone hormonal activity are provided. The hybrid polypeptides of theinvention comprise at least two bio-active peptide hormone modulescovalently linked together, wherein at least one of the bio-activepeptide hormone modules exhibits at least one hormonal activity of acomponent peptide hormone. The bio-active peptide hormone modules areindependently selected from: component peptide hormones, fragments ofcomponent peptide hormones that exhibit at least one hormonal activityof the component peptide hormones, analogs and derivatives of componentpeptide hormones that exhibit at least one hormonal activity of thecomponent peptide hormones, fragments of analogs and derivatives ofcomponent peptide hormones that exhibit at least one hormonal activityof the component peptide hormones, and peptidic enhancers.

In one embodiment a hybrid polypeptide is exhibiting at least onehormonal activity, the hybrid polypeptide containing at least a firstbio-active peptide hormone module covalently linked to at least oneadditional bio-active peptide hormone module; wherein the bio-activepeptide hormone modules are independently selected from the groupconsisting of: component peptide hormones; fragments of componentpeptide hormones that exhibit at least one hormonal activity of thecomponent peptide hormones; analogs and derivatives of component peptidehormones that exhibit at least one hormonal activity of the componentpeptide hormones; fragments of analogs and derivatives of componentpeptide hormones that exhibit at least one hormonal activity of thecomponent peptide hormones; and peptidic enhancers. The componentpeptide hormones are typically independently selected from at least twoof the group consisting of amylin, adrenomedullin (ADM), calcitonin(CT), calcitonin gene related peptide (CGRP), intermedin,cholecystokinin (“CCK”), leptin, peptide YY (PYY), glucagon-likepeptide-1 (GLP-1), glucagon-like peptide 2 (GLP-2), oxyntomodulin (OXM),a natriuretic peptide, and exendin-4. Typically peptidic enhancers areindependently selected from the group consisting of structural motifs ofcomponent peptide hormones that impart a desired chemical stability,conformational stability, metabolic stability, bioavailability,organ/tissue targeting, receptor interaction, protease inhibition,plasma protein binding, or other pharmacokinetic characteristic to thehybrid polypeptide, and structural motifs of analogs or derivatives ofcomponent peptide hormones that impart a desired chemical stability,conformational stability, metabolic stability, bioavailability,organ/tissue targeting, receptor interaction, protease inhibition,plasma protein binding, or other pharmacokinetic characteristic to thehybrid polypeptide. In yet a further embodiment at least one of thebio-active peptide hormone modules exhibits at least one hormonalactivity of a component peptide hormone. In yet further alternativeembodiments when the at least one bio-active peptide hormone module thatexhibits at least one hormonal activity of a component peptide hormoneis amylin, a fragment of amylin that exhibits at least one hormonalactivity, an analog or derivative of amylin that exhibits at least onehormonal activity, or a fragment of an analog or derivative of amylinthat exhibits at least one hormonal activity, and the at least one otherbio-active peptide hormone module is CCK, a fragment of CCK thatexhibits at least one hormonal activity, an analog or derivative of CCKthat exhibits at least one hormonal activity, a fragment of an analog orderivative of CCK that exhibits at least one hormonal activity, CT, afragment of CT that exhibits at least one hormonal activity, an analogor derivative of CT that exhibits at least one hormonal activity, or afragment of an analog or derivative of CT that exhibits at least onehormonal activity, then the hybrid polypeptide can further contain atleast three bio-active peptide hormone modules selected from at leastthree different component peptide hormones. In yet a further alternativeembodiment, when the at least one bio-active peptide hormone module thatexhibits at least one hormonal activity of a component peptide hormoneis GLP-1, a fragment of GLP-1 that exhibits at least one hormonalactivity, an analog or derivative of GLP-1 that exhibits at least onehormonal activity, or a fragment of an analog or derivative of GLP-1that exhibits at least one hormonal activity, and the at least one otherbio-active peptide hormone module is a peptidic enhancer comprising anexendin fragment, then the hybrid polypeptide can further contain atleast three bio-active peptide hormone modules.

Component peptide hormones of the invention include: amylin,adrenomedullin (ADM), calcitonin (CT), calcitonin gene related peptide(CGRP), intermedin, cholecystokinin (“CCK”), leptin, peptide YY (PYY),glucagon-like peptide-1 (GLP-1), glucagon-like peptide 2 (GLP-2),oxyntomodulin (OXM), natriuretic peptides, and exendin-4;

Peptidic enhancers of the invention include: structural motifs ofcomponent peptide hormones that impart a desired chemical stability,conformational stability, metabolic stability, bioavailability,organ/tissue targeting, receptor interaction, protease inhibition,plasma protein binding, or other pharmacokinetic characteristic to thehybrid polypeptide, and structural motifs of analogs or derivatives ofcomponent peptide hormones that impart a desired chemical stability,conformational stability, metabolic stability, bioavailability,organ/tissue targeting, receptor interaction, protease inhibition,plasma protein binding, or other pharmacokinetic characteristic to thehybrid polypeptide.

In another aspect of the invention, methods for treating or preventingobesity are provided, wherein the method comprises administering atherapeutically or prophylactically effective amount of a hybridpolypeptide of the invention to a subject in need thereof. In apreferred embodiment, the subject is an obese or overweight subject.While “obesity” is generally defined as a body mass index over 30, forpurposes of this disclosure, any subject, including those with a bodymass index of less than 30, who needs or wishes to reduce body weight isincluded in the scope of “obese.” Subjects who are insulin resistant,glucose intolerant, or have any form of diabetes mellitus (e.g., type 1,2 or gestational diabetes) can benefit from this method.

In yet another aspect of the invention, methods of reducing food intake,reducing nutrient availability, causing weight loss, treating diabetesmellitus or diabetes-associated conditions, and improving lipid profile(including reducing LDL cholesterol and triglyceride levels and/orchanging HDL cholesterol levels) are provided, wherein the methodscomprise administering to a subject an effective amount of a hybridpolypeptide of the invention. In a preferred embodiment, the methods ofthe invention are used to treat or prevent conditions or disorders whichcan be alleviated by reducing nutrient availability in a subject in needthereof, comprising administering to said subject a therapeutically orprophylactically effective amount of a hybrid polypeptide of theinvention. In another embodiment, the methods of the invention are usedto treat or prevent conditions or disorders which can be alleviated bycontrol plasma glucose levels, insulin levels, and/or insulin secretion.In yet another embodiment, the methods of the invention are used totreat diabetes and/or diabetes-related conditions. Such conditions anddisorders include, but are not limited to, hypertension, dyslipidemia,cardiovascular disease, eating disorders, insulin-resistance, obesity,and diabetes mellitus of any kind, including Type I, Type II, andgestational diabetes, diabetes complications (neuropathy (based on,e.g., neurotrophic actions of exendin-4), neuropathic pain (based on,e.g., amylin action), retinopathy, nephropathy, conditions ofinsufficient pancreatic beta cell mass (based on, e.g., islet neogenesisactions of exendin-4 and GLP-1).

The present invention also relates to pharmaceutical compositionscomprising a therapeutically or prophylactically effective amount of atleast one hybrid polypeptide of the invention, or a pharmaceuticallyacceptable salt thereof, together with pharmaceutically acceptablediluents, preservatives, solubilizers, emulsifiers, adjuvants and/orcarriers useful in the delivery of the hybrid polypeptides.

These and other aspects of the invention will be more clearly understoodwith reference to the following preferred embodiments and detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the effect of exemplary compounds of the inventionin DIO mouse assay.

FIG. 2 demonstrates the effect of exemplary compounds of the inventionin DIO mouse assay.

FIGS. 3A-3C demonstrate the effect of exemplary compounds of theinvention in DIO mouse assay.

FIGS. 4A-4B demonstrate the effects of exemplary compounds of theinvention in food intake assay, compared to parent peptide compounds.

FIGS. 5A-5B demonstrate the effects of exemplary compounds of theinvention in blood glucose lowering assay and food intake assay,respectively.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates generally to novel, selectable hybridpolypeptides useful as agents for the treatment and prevention ofmetabolic diseases and disorders which can be alleviated by control ofplasma glucose levels, insulin levels, and/or insulin secretion, such asdiabetes and diabetes-related conditions. Such conditions and disordersinclude, but are not limited to, hypertension, dyslipidemia,cardiovascular disease, eating disorders, insulin-resistance, obesity,and diabetes mellitus of any kind, including type 1, type 2, andgestational diabetes.

In one aspect, the invention involves the modular assembly ofphysiologically, metabolically, and/or pharmacokinetically activepeptidic modules that may be selectable based on “bio-activities”, e.g.,therapeutic efficacy, scope of function, duration of action,physicochemical properties, and/or other pharmacokinetic properties.

Without intending to be limited by theory, the present invention relatesat least in part to a “toolbox” approach, wherein bio-active peptidehormone modules are linked in binary, tertiary or higher ordercombinations to create novel, efficacious therapeutic agents withselectable properties. The “bio-active peptide hormone modules” may bepeptide hormones, peptide fragments with hormonal activity, orstructural motifs of peptide hormones that impart chemical, metabolic,and/or other pharmacokinetic stability. The peptide hormones can includenative peptide hormones, as well as peptide hormone analogs andderivatives, as known in the art and described herein.

In one aspect of the invention, it has been found that the combinationof certain physicochemical characteristics of two or more peptidehormones into a single modality can facilitate intervention at severalpoints in a dysfunctional metabolic circuit. As such, in one aspect ofthe invention, rationally-designed hybrid polypeptides are provided thatintegrate selectable bio-activities into a single polypeptide agent. Inone embodiment, the selectable hybrid polypeptides of the invention mayinvolve the use of chemically stable linkers to covalently attach thebio-active modules. In another embodiment, the selectable hybridpolypeptides of the invention may involve the use of cleavable linkers,which themselves may be or form part of a bio-active module.

Again, without intending to be limited by theory, design of the hybridpolypeptides of the present invention may generally involve: (1) theidentification, selection and pairing of bio-active peptide hormonemodules for desired efficacy and therapeutic use, and (2) the covalentlinking of the bio-active modules (e.g. native peptide hormones, peptidehormone analogs or derivatives with hormonal activity, peptide hormonefragments with hormonal activity, stabilizing motifs, etc.) eitherdirectly or via a linker without loss of bio-activity of the componentmodules. In certain embodiments, module selection criteria may include,but not be limited to: (a) desired in vivo efficacy for desiredtherapeutic or prophylactic indication, such as an additive or asynergistic effect; (b) optional synergism or dual action of the linkedmodules for multiple therapeutic or prophylactic indications; and/or (c)a desired chemical stability, conformational stability, metabolicstability, bioavailability, organ/tissue targeting, receptorinteraction, protease inhibition, plasma protein binding, and/or otherpharmacokinetic characteristic.

The section headings are used herein for organizational purposes only,and are not to be construed as in any way limiting the subject matterdescribed.

Hybrid Polypeptides of the Invention

As mentioned above, the present invention relates in part to hybridpolypeptides comprising at least two bio-active peptide hormone modulesselectable from component peptide hormones described herein. The hybridpolypeptides of the present invention will generally be useful in thetreatment and prevention of metabolic conditions and disorders. Thehybrid polypeptides of the invention will exhibit at least one hormonalactivity of a component peptide hormone, and may preferably include atleast one additional bio-activity of a second component peptide hormone.

In one embodiment, the hybrid polypeptides of the invention may compriseat least two bio-active peptide hormone modules, wherein each of said atleast two bio-active peptide hormone modules exhibits at least onehormonal activity of a component peptide hormone. In another embodiment,the hybrid polypeptides of the invention may comprise at least twobio-active peptide hormone modules, wherein at least one of saidbio-active peptide hormone modules exhibits at least one hormonalactivity of a component peptide hormone and at least one of saidbio-active peptide hormone modules imparts a desired chemical stability,conformational stability, metabolic stability, bioavailability,organ/tissue targeting, receptor interaction, protease inhibition,plasma protein binding, and/or other pharmacokinetic characteristic tothe hybrid polypeptide.

In a preferred embodiment, the hybrid polypeptides of the invention mayhave comparable or higher potency in the treatment and/or prevention ofmetabolic conditions and disorders, as compared to the component peptidehormones. In another embodiment, the hybrid polypeptides of theinvention may have comparable or higher potency in the treatment and/orprevention of diabetes and/or diabetes-related disorders, as compared tothe component peptide hormones. Alternatively, preferred hybridpolypeptides of the invention may exhibit improved ease of manufacture,stability, and/or ease of formulation, as compared to the componentpeptide hormones.

More particularly, the hybrid polypeptides of the present invention willgenerally comprise a first bio-active peptide hormone module covalentlylinked to at least one additional bio-active peptide hormone module. Thebio-active peptide hormone modules may be covalently linked together inany manner known in the art, including but not limited to direct amidebonds or chemical linker groups, as described in further detail herein.In one embodiment, chemical linker groups may include peptide mimeticswhich induce or stabilize polypeptide conformation.

The first bio-active peptide hormone module may be selected from a firstcomponent peptide hormone, and may be a peptide hormone (includingnative peptide hormones as well as analogs and derivatives thereof), apeptide fragment with hormonal activity (including fragments of nativepeptides hormones as well as analogs and derivatives thereof), or astructural motif of a peptide hormone (including native peptide hormonesas well as analogs and derivatives thereof) that imparts a desiredchemical stability, conformational stability, metabolic stability,bioavailability, organ/tissue targeting, receptor interaction, proteaseinhibition, plasma protein binding, and/or other pharmacokineticcharacteristic to the hybrid polypeptide. Likewise, the additionalbio-active peptide module(s) may be selected from component peptidehormones, and may be a peptide hormone (including native peptidehormones as well as analogs and derivatives thereof), a peptide fragmentwith hormonal activity (including fragments of native peptides hormonesas well as analogs and derivatives thereof), or a structural motif of ahormone peptide (including native peptide hormones as well as analogsand derivatives thereof) that imparts a desired chemical stability,conformational stability, metabolic stability, bioavailability,organ/tissue targeting, receptor interaction, protease inhibition,plasma protein binding, and/or other pharmacokinetic characteristic tothe hybrid polypeptide. The first peptide hormone and the additionalpeptide hormone may be the same peptide hormone, may be from the samefamily of peptide hormones, or may be different peptide hormones,depending on the desired characteristics of the bio-active peptidehormone modules.

As used herein, the term “bio-active” refers to (1) biological activityin at least one in vivo hormonal pathway, or (2) modulation of thetherapeutic efficacy, scope of function, duration of action,physicochemical properties, and/or other pharmacokinetic properties ofsuch biological activity. Biological activity may be evaluated throughtarget hormone receptor binding assays, or through metabolic studiesthat monitor a physiological indication, as known in the art anddescribed herein. Modulation of the therapeutic efficacy, scope offunction, duration of action, physicochemical properties, and/or otherpharmacokinetic properties of such biological activity may be modifiedthrough changed in, e.g., chemical stability, conformational stability,metabolic stability, bioavailability, organ/tissue targeting, receptorinteraction, protease inhibition, plasma protein binding, and/or otherpharmacokinetic characteristics.

In one embodiment, the hybrid polypeptides of the invention retain atleast about 25%, preferably about 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 98%, or 99% percent of the biological activity of a componentpeptide hormone. Preferred hybrid polypeptides are those having apotency in one of the metabolic-related assays known in the art ordescribed herein (e.g., receptor binding, food intake, gastric emptying,pancreatic secretion, insulin secretion, blood glucose lowering, weightreduction, etc.) which is equal to or greater than the potency ofcomponent peptide hormone in that same assay. Alternatively, preferredhybrid polypeptides of the invention may exhibit improved ease ofmanufacture, stability, and/or ease of formulation, as compared tocomponent peptide hormones.

In another embodiment, the hybrid polypeptides of the invention retainat least about 25%, preferably about 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 98%, or 99% percent of the biological activity of a nativecomponent peptide hormone with regard to the reduction of nutrientavailability, the reduction of food intake, the effect of body weightgain, and/or the treatment and prevention of metabolic conditions anddisorders. In yet another embodiment, the hybrid polypeptides of theinvention exhibit at least about 110%, 125%, 130%, 140%, 150%, 200%, ormore of the biological activity of a native peptide hormone with regardto the reduction of nutrient availability the reduction of food intake,the effect of body weight gain, and/or the treatment and prevention ofmetabolic conditions and disorders. In another embodiment, the hybridpolypeptides of the invention exhibit improved component peptide hormonereceptor agonist activity.

1. Component Peptides Hormones, Analogs and Derivatives

Component peptide hormones generally include peptide hormones useful inthe treatment or prevention of metabolic diseases and disordersincluding: (a) the amylin family, including amylin, adrenomedullin(“ADM”), calcitonin (“CT”), calcitonin gene related peptide (“CGRP”),intermedin (also known as “AFP-6”) and related peptides; (b)cholecystokinin (“CCK”); (c) the leptin family, including leptin andleptin-like peptides; (d) the pancreatic polypeptide family, includingpancreatic polypeptide (“PP”) and peptide YY (“PYY”); (e) incretins andincretin mimetics, including: peptide hormones derived from theproglucagon gene such as: glucagon, glucagon-like peptide-1 (“GLP-1”),glucagon-like peptide 2 (“GLP-2”), and oxyntomodulin (“OXM”); andexendins such as: exendin-3, and exendin-4; and (f) natriuretic peptidesincluding ANP, BNP, CNP, and urodilatin, their precursor forms andpeptides derived therefrom (g) urocortin family and the (h) neuromedinfamily, and analogs, derivatives and fragments thereof. As discussedherein component peptide hormones of the invention also include analogsand derivatives that retain hormonal activity of these native peptidehormones. In one embodiment, such analogs and derivatives are agonistsof the target hormone receptor.

By “amylin” is meant the human peptide hormone referred to as amylin andsecreted from the beta cells of the pancreas, and species variationsthereof, as described in U.S. Pat. No. 5,234,906, issued Aug. 10, 1993,for “Hyperglycemic Compositions,” the contents of which are herebyincorporated by reference. More particularly, amylin is a 37-amino acidpolypeptide hormone normally co-secreted with insulin by pancreatic betacells in response to nutrient intake (see, e.g., Koda et al., Lancet339:1179-1180, 1992). In this sense, “amylin,” “wild-type amylin,” and“native amylin,” i.e., unmodified amylin, are used interchangeably.

By “adrenomedullin” or “ADM” is meant the human peptide hormone andspecies variants thereof. More particularly, ADM is generated from a 185amino acid preprohormone through consecutive enzymatic cleavage andamidation. This process culminates in the liberation of a 52 amino acidbioactive peptide.

By “calcitonin” or “CT” is meant the human peptide hormone and speciesvariants thereof, including salmon calcitonin (“sCT”). Moreparticularly, CT is a 32 amino acid peptide cleaved from a largerprohormone. It contains a single disulfide bond, which causes the aminoterminus to assume the shape of a ring. Alternative splicing of thecalcitonin pre-mRNA can yield a mRNA encoding calcitonin gene-relatedpeptide; that peptide appears to function in the nervous and vascularsystems. The calcitonin receptor has been cloned and shown to be amember of the seven-transmembrane, G protein-coupled receptor family.

By “calcitonin gene related peptide” or “CGRP” is meant the humanpeptide hormone and species variants thereof, in any physiological form.

By “intermedin” or “AFP-6” is meant the human peptide hormone andspecies variants thereof, in any physiological form.

By “cholecystokinin” or “CCK” is meant the human peptide hormone andspecies variants thereof. More particularly, CCK is a 33-amino acidsequence first identified in humans, and includes a 8-amino acid in vivoC-terminal fragment (“CCK-8”) that has been reportedly demonstrated inpig, rat, chicken, chinchilla, dog and humans. Thus, the term CCK-33will generally refer to human CCK(1-33), while CCK-8 (CCK(26-33); SEQ IDNO:55) will refer to the C-terminal octapeptide generically in both thesulfated and unsulfated unless otherwise specified. Further,pentagastrin or CCK-5 will refer to the C-terminal peptide CCK(29-33)(SEQ ID NO:209), and the CCK-4 will refer to the C-terminal tetrapeptideCCK(30-33) (SEQ ID NO:208). However, as used herein, CCK will generallyrefer to all naturally occurring variations of the hormone, includingCCK-33, CCK-8, CCK-5, and CCK-4, in the sulfated and unsulfated formunless otherwise specified.

By “leptin” is meant the naturally occurring leptin from any species, aswell as biologically active D-isoforms, or fragments of naturallyoccurring leptin and variants thereof, and combinations of thepreceding. Leptin is the polypeptide product of the ob gene as describedin the International Patent Publication No. WO 96/05309, which isincorporated herein by reference in its entirety. Putative analogs andfragments of leptin are reported in U.S. Pat. No. 5,521,283, U.S. Pat.No. 5,532,336, PCT/US96/22308 and PCT/US96/01471, each of which isincorporated herein by reference in its entirety.

By “PP” is meant human pancreatic peptide polypeptide or speciesvariants thereof, in any physiological form. Thus, the term “PP”includes both the human full length, 36 amino acid peptide as set forthin SEQ ID NO: 290, and species variations of PP, including, e.g.,murine, hamster, chicken, bovine, rat, and dog PP. In this sense, “PP,”“wild-type PP,” and “native PP,” i.e., unmodified PP, are usedinterchangeably.

By “PYY” is meant human peptide YY polypeptide or species variantsthereof, in any physiological form. Thus, the term “PYY” includes boththe human full length, 36 amino acid peptide, and species variations ofPYY, including e.g., murine, hamster, chicken, bovine, rat, and dog PYY.In this sense, “PYY” and “wild-type PYY” and “native PYY,” i.e.,unmodified PYY, are used interchangeably. In the context of the presentinvention, all modifications discussed with reference to the PYY analogpolypeptides of the present invention are based on the 36 amino acidsequence of native human PYY.

By “GLP-1” is meant human glucagon like peptide-1 or species variantsthereof, in any physiological form. The term “GLP-1” includes humanGLP-1(1-37) (SEQ ID NO:59), GLP-1(7-37) (SEQ ID NO:204), andGLP-1(7-36)amide (SEQ ID NO:61), with reference to the full length humanGLP-1(1-37) (SEQ ID NO:59), and species variations of GLP-1, including,e.g., murine, hamster, chicken, bovine, rat, and dog PP. In this sense,“GLP-1,” “wild-type GLP-1,” and “native GLP-1,” i.e., unmodified GLP-1,are used interchangeably.

By “GLP-2” is meant human glucagon like peptide-2 or species variantsthereof, in any physiological form. More particularly, GLP-2 is a 33amino acid peptide, co-secreted along with GLP-1 from intestinalendocrine cells in the small and large intestine.

By “OXM” is meant human oxyntomodulin or species variants thereof in anyphysiological form. More particularly, OXM is a 37 amino acid peptidethat contains the 29 amino acid sequence of glucagon followed by an 8amino acid carboxyterminal extension.

By “exendin” is meant a peptide hormone found in the saliva of theGila-monster, a lizard endogenous to Arizona, and the Mexican BeadedLizard, as well as species variants thereof. More particularly,Exendin-3 is present in the saliva of Heloderma horridum, and exendin-4is present in the saliva of Heloderma suspectum (Eng, J., et al., J.Biol. Chem., 265:20259-62, 1990; Eng., J., et al., J. Biol. Chem.,267:7402-05 (1992)). The exendins have some sequence similarity toseveral members of the glucagon-like peptide family, with the highestidentity, 53%, being to GLP-1 (Goke, et al., J. Biol. Chem.,268:19650-55 (1993)). In this sense, “exendin,” “wild-type exendin,” and“native exendin,” i.e., unmodified exendin, are used interchangeably.

By “urocortin” is meant a human urocortin petide hormone or speciesvariants thereof in any physiological form. More particularly, there arethree human urocortins: Ucn-1, Ucn-2 and Ucn-3. For example, humanurocortin 1 has the formula:Asp-Asn-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val-NH2 (SEQ ID NO:294). Rat-derived urocortin is identical but for 2substitutions: Asp2 for Asn2 and Pro4 for Ser4. Human Ucn-2 has thesequence Ile Val Leu Ser Leu Asp Val Pro Ile Gly Leu Leu Gln Ile Leu LeuGlu Gln Ala Arg Ala Arg Ala Ala Arg Glu Gln Ala Thr Thr Asn Ala Arg IleLeu Ala Arg Val Gly His Cys (SEQ ID NO:399). Human Ucn-3 has thesequence Phe Thr Leu Ser Leu Asp Val Pro Thr Asn Ile Met Asn Leu Leu PheAsn Ile Ala Lys Ala Lys Asn Leu Arg Ala Gln Ala Ala Ala Asn Ala His LeuMet Ala Gln Ile (SEQ ID NO:299). Ucn-3 is preferably in amide form.Further urocortins and analogs are described in the literature, forexample in U.S. Pat. No. 6,214,797. Urocortins Ucn-2 and Ucn-3, whichretain the food-intake suppression andantihypertensive/cardioprotective/inotropic properties, find particularuse in the hybrids of the ivention. Stresscopin (Ucn-3) andStresscopin-related peptide (Ucn 2), named for their ability to suppressthe chronic HPA activation following a stressful stimulus such asdieting/fasting, are specific for the CRF type 2 receptor and do notactivate CRF-R1 which mediates ACTH release. Hybrids comprising aurocortin, e.g., Ucn-2 or Ucn-3, are particularly useful forvasodilation and thus for cardiovascular uses as described herein, e.gCHF. Urocortin containing hybrids of the invention find particular usein treating or preventing conditions associated with stimulating ACTHrelease, hypertension due to vasodilatory effects, inflammation mediatedvia other than ACTH elevation, hyperthermia, appetite disorder,congestive heart failure, stress, anxiety, and psoriasis. Such compoundsare also useful for an antiproliferative effect, such as for treating orpreventing cancers or tumor growth. Of particular interest are urocortinpeptide hormone module combined with a natriuretic peptide module,amylin family, aan exendin family, or a GLP1 family module to provide anenhanced cardiovascular benefit, e.g. treating CHF, as by providing abeneficial vasodilation effect.

By “neuromedin” is meant the neuromedin family of peptides includingneuromedin U and S peptides, more particularly their active hormonesequences. For example, the native active human nueromedin U peptidehormone is neuromedin-U25: Phe Arg Val Asp Glu Glu Phe Gln Ser Pro PheAla Ser Gln Ser Arg Gly Tyr Phe Leu Phe Arg Pro Arg Asn (SEQ ID NO:308),particularly in the amide form. Pig U25 has the sequence:FKVDEEFQGPIVSQNRRYFLFRPRN (SEQ ID NO:314), particularly its amide form.Other neuromedin U family members include the following listed as theirSWISS-PROT designations and entry numbers: NEUU_CANFA (P34962),NEUU_CAVPO (P34966), NEUU_CHICK (P34963), NEUU_HUMAN (P48645),NEUU_LITCE (P81872), NEUU_MOUSE (Q9QXK8), NEUU_PIG (P34964), NEUU_RABIT(P34965), NEUU_RANTE (P20056), and NEUU_RAT (P12760). Of particularinterest are their processed active peptide hormones and analogs,derivatives and fragments thereof. Included in the neuromedin U familyare various truncated or splice varinats, e.g.,FLFHYSKTQKLGKSNVVEELQSPFASQSRGYFLFRPRN (SEQ ID NO:300). Exemplary of theneuromedin S family is human neuromedin S with the sequenceILQRGSGTAAVDFTKKDHTATWGRPFFLFRPRN (SEQ ID NO:315), particularly itsamide form. Hybrids of the invention having neuromedin module will ananorexigenic effect, and thus have beneficial value in treating obesity,diabetes, reducing food intake, and other related conditions anddisorders as described herein. Of particular interest are neuromedinmodules combined with an amylin family peptide, an exendin peptidefamily or a GLP1 peptide family module.

As used herein, an “analog” refers to a peptide whose sequence wasderived from that of a base reference peptide (e.g., PP, PYY, amylin,GLP-1, exendin, etc.), including insertions, substitutions, extensions,and/or deletions of the reference amino acid sequence, preferably havingat least 50 or 55% amino acid sequence identity with the base peptide,more preferably having at least 70%, 80%, 90%, or 95% amino acidsequence identity with the base peptide. In one embodiment, such analogsmay comprise conservative or non-conservative amino acid substitutions(including non-natural amino acids and L and D forms).

A “derivative” is defined as a molecule having the amino acid sequenceof a native reference peptide or analog, but additionally havingchemical modification of one or more of its amino acid side groups,α-carbon atoms, terminal amino group, or terminal carboxylic acid group.A chemical modification includes, but is not limited to, adding chemicalmoieties, creating new bonds, and removing chemical moieties.Modifications at amino acid side groups include, without limitation,acylation of lysine s-amino groups, N-alkylation of arginine, histidine,or lysine, alkylation of glutamic or aspartic carboxylic acid groups,and deamidation of glutamine or asparagine. Modifications of theterminal amino include, without limitation, the desamino, N-lower alkyl,N-di-lower alkyl, constrained alkyls (e.g. branched, cyclic, fused,adamantyl) and N-acyl modifications. Modifications of the terminalcarboxy group include, without limitation, the amide, lower alkyl amide,constrained alkyls (e.g. branched, cyclic, fused, adamantyl) alkyl,dialkyl amide, and lower alkyl ester modifications. Lower alkyl is C1-C4alkyl. Furthermore, one or more side groups, or terminal groups, may beprotected by protective groups known to the ordinarily-skilled peptidechemist. The α-carbon of an amino acid may be mono- or dimethylated.

By “agonist” is meant a compound which elicits a biological activity ofnative human reference peptide, preferably having a potency better thanthe reference peptide, or within five orders of magnitude (plus orminus) of potency compared to the reference peptide, more preferably 4,3, 2, or 1 order of magnitude, when evaluated by art-known measures suchas receptor binding/competition studies. In one embodiment, the termsrefer to a compound which elicits a biological effect similar to that ofnative human reference peptide, for example a compound (1) havingactivity in the food intake, gastric emptying, pancreatic secretion, orweight loss assays similar to native human reference peptide, or (2)which binds specifically in a reference receptor assay or in acompetitive binding assay with labeled reference peptide. Preferably,the agonists will bind in such assays with an affinity of greater than 1μM, and more preferably with an affinity of greater than 1-5 nM. Inanother embodiment, the terms refer to a compound which elicits abiological effect in the treatment of diabetes or a diabetes relatedcondition or disorder. Such agonists may comprise a polypeptidecomprising an active fragment of a reference peptide or a small chemicalmolecule.

By “amino acid” and “amino acid residue” is meant natural amino acids,unnatural amino acids, and modified amino acid. Unless stated to thecontrary, any reference to an amino acid, generally or specifically byname, includes reference to both the D and the L stereoisomers if theirstructure allow such stereoisomeric forms. Natural amino acids includealanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp),cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly),histidine (His), isoleucine (Ile), leucine (Leu), Lysine (Lys),methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser),threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val).Unnatural amino acids include, but are not limited to homo-lysine,homo-arginine, azetidinecarboxylic acid, 2-aminoadipic acid,3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutyricacid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid,2aminoisobutyric acid, 3-aminoisbutyric acid, 2-aminopimelic acid,tertiary-butylglycine, 2,4-diaminoisobutyric acid, desmosine,2,2′-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine,N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine,3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine,N-methylalanine, N-methylglycine, N-methylisoleucine,N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline,norleucine, ornithine, pentylglycine, pipecolic acid and thioproline.Additional unnatural amino acids include modified amino acid residueswhich are chemically blocked, reversibly or irreversibly, or chemicallymodified on their N-terminal amino group or their side chain groups, asfor example, N-methylated D and L amino acids or residues wherein theside chain functional groups are chemically modified to anotherfunctional group. For example, modified amino acids include methioninesulfoxide; methionine sulfone; aspartic acid-(beta-methyl ester), amodified amino acid of aspartic acid; N-ethylglycine, a modified aminoacid of glycine; or alanine carboxamide, a modified amino acid ofalanine. Additional residues that can be incorporated are described inSandberg et al., J. Med. Chem. 41: 2481-91, 1998.

As used herein: “5 Apa” means 5 amino-pentanoyl, “12 Ado” means 12-aminododecanoyl, “PEG(8)” mean 3,6,-dioxyoctanoyl, and “PEG(13)” means1-amino-4,7,10-trioxa-13-tridecanamine succinimoyl.

As discussed herein native component peptide hormones are known in theart, as are their analogs and derivatives. For reference, the sequencesof several native component peptide hormones are provided in Table 1.

TABLE 1 Exemplary Component Peptide Hormones Seq ID Description Sequence44 Rat Amylin KCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTY 45 h-AmylinKCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY 46 h-ADMYRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAP RSKISPQGY 47 s-CTCSNLSTCVLGKLSQELHKLQTYPRTNTGSGTP 48 h-CTCGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP 49 h-CGRP αACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF 50 h-CGRP βACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSKAF 51 h-AFP-6 (1-TQAQLLRVGCVLGTCQVQNLSHRLWQLMGPAGRQDSAPVDPS 47) SPHSY 52 h-AFP-6 (8-VGCVLGTCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY 47) 53 Mouse AFP-PHAQLLRVGCVLGTCQVQNLSHRLWQLVRPAGRRDSAPVDPSS 6 (1-47) PHSY 54 Mouse AFP-VGCVLGTCQVQNLSHRLWQLVRPAGRRDSAPVDPSSPHSY 6 (8-47) 55 CCK-8-DY(SO₃)MGWMDF sulfated 56 h-LeptinMHWGTLCGFLWLWPYLFYVQAVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC 57 HPYYYPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY 58 HPYY(3-36)IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY 59 hGLP-1 (1-HDEFERHAEGTFTSDVSSTLEGQAALEFIAWLVKGRG 37) 60 Frog GLP-1HAEGTYTNDVTEYLEEKAAKEFIEWLIKGKPKKIRYS-OH; 188 Frog GLP-1HAEGTFTSDVTQQLDEKAAKEFIDWLINGGPSKEIIS-OH 61 h-GLP-1 (7-HAEGTFTSDVSSYLEGQAALEFIAWLVKGR 36) 62 h-GLP-2HADGSFSDEMNTILDNLAARDFINWLIETKITD 63 Frog GLP-2HAEGTFTNDMTNYLEEKAAKEFVGWLIKGRP-OH 64 OXMHSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA 65 Exendin-3HSDGTFTSDLSKQMEEEAVR LFIEWLKNGG PSSGAPPPS 66 Exendin-4HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS 291 Urocortin IIVILSLDVPIGLLRILLEQARYKAARNQAATNAQILAHV-NH2 (Mouse) 292 WP-24WSPGARNQGGGARALLLLLAERFP-OH (Urocortin) 293 TV-18 TQSQRERAEQNRIIFDSV-NH2(Urocortin) 294 Human DNPSLSIDLTFHLLRTLLELARTQSQRERAEQNRIIFDSV-NH2Urocortin 295 SE-20 SFHYLRSRDASSGEEEEGKE-OH (Urocortin- 111/Stresscopin)296 AI-13 AQAAANAHLMAQI-OH (Urocortin- III/Stresscopin) 297 DA-21DNPSLSIDLTFHLLRTLLELA-OH (Urocortin) 298 TL-26TKFTLSLDVPTNIMNLLFNIAKAKNL-OH (Urocortin- III/ Stresscopin) 299 HumanFTLSLDVPTNIMNLLFNIAKAKNLRAQAAANAHLMAQI-NH2 Urocortin III 300 FN-38FLFHYSKTQKLGKSNVVEELQSPFASQSRGYFLFRPRN-NH2 (SLM14) 301 alpha-AtrialSLRRSSCFGGRMDRIGAQSGLGCNSFRY-OH Natriuretic Polypeptide (1-28) human,porcine, bovive 302 Brain c(NSKMAHSSSCFGQKIDRIGAVSRLGCDGLRLF)-OHnatriuretic peptide, Rat; BNP, Rat 303 BrainSPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH-OH Natriuretic Peptide (BNP) (human)304 C-TYPE GLSKGCFGLKLDRIGSMSGLGC-OH Natriuretic peptide, Porcine; Cnp,Porcine 305 Neuromedin YFLFRPRN-NH2 U-8 (porcine) 306 Neuromedin YKVNEYQGPVAPSGGFFLFRPRN-NH2 U (rat) 307 Neuromedin  GYFLFRPRN-NH2 U-9308 Neuromedin  FRVDEEFQSPFASQSRGYFLFRPRN-NH2 (U25), human

These peptides are generally C-terminally amidated when expressedphysiologically, but need not be for the purposes of the instantinvention. In other words, the C-terminus of these peptides, as well asthe hybrid polypeptides of the present invention, may have a free —OH or—NH₂ group. These peptides may also have other post-translationalmodifications. One skilled in the art will appreciate that the hybridpolypeptides of the present invention may also be constructed with anN-terminal methionine residue.

Exemplary peptide modules for use in the invention further include,N-terminally extendable peptide modules (and their analogs andfragments) including Apelin, which exists in 2 forms, Apelin 36 and 13,both active at the AJP receptor (LVQPRGSRNGPGPWQGGRRKFRRQRPRLSHKGPMPF-OH(SEQ ID NO:316) and pERPRLSHKGPMPF-OH) (SEQ ID NO:317); ProlactinReleasing peptide, which exists in 2 forms, PRP31 and PRP20, equallyactive at GPR10 (SRTHRHSMEIRTPDINPAWYASRGIRPVGRF-NH2 (SEQ ID NO:318) andTPDINPAWYASRGIRPVGRF-NH2) (SEQ ID NO:319); Gastrin, which exists as biggastrin and mini-gastrin, the bulk of activity however residing inresides in pentagastrin (QLGPQGPPHLVADPSKKQGPWLEEEEEAYGWMDF-NH2; (SEQ IDNO:320); pEGPWLEEEEEAYGWMDF-NH2 (SEQ ID NO:321); beta-AWMDF-NH2 (SEQ IDNO:322)); CCK, which exists as CCK33 or CCK8 (central vs. peripheral;KAPSGRMSIVKNLQNLDPSHRISDRDYMGWMDF-NH2 (SEQ ID NO:323); DYMGWMDF-NH2)(SEQ ID NO:55); Cortistatin, which exists as cortistatin 17 or 29(QEGAPPQQSARRDRMPCRNFFWKTFSSCK-OH (SEQ ID NO:324) andDRMPCRNFFWKTFSSCK-OH) (SEQ ID NO:325); somatostatin, which exists assomatostatin 14 or 28 (SANSNPAMAPRERKAGCKNFFWKTFTSC-OH (SEQ ID NO:326);AGCKNFFWKTFTSC-OH (SEQ ID NO:327)); GRP for which a C-terminal 10 aminoacid sequence possesses most of the activity(VPLPAGGGTVLTKMYPRGNHWAVGHLM-NH2 (SEQ ID NO:328); GNHWAVGHLM-NH2 (SEQ IDNO:329)); Neuromedin B for which a C-terminal 10 amino acid regionpossesses most of the activity (LSWDLPEPRSRASKIRVHSRGNLWATGHFM-NH2 (SEQID NO:330); GNLWATGHFM-NH2 (SEQ ID NO:331)); Neuromedin S for which aC-terminal 9 amino acid region possesses most of the activity(ILQRGSGTAAVDFTKKDHTATWGRPFFLFRPRN-NH2 (SEQ ID NO:315); PFFLFRPRN-NH2(SEQ ID NO:332)); Neuromedin U for which a C-terminal 9 amino acidregion possesses most of the activity (FRVDEEFQSPFASQSRGYFLFRPRN-NH2(SEQ ID NO:308); GYFLFRPRN-NH2 (SEQ ID NO:307)); Neurotensin, whichexists as long and short forms (KIPYILKRQLYENKPRRPYIL-OH (SEQ IDNO:333); QLYENKPRRPYIL-OH) (SEQ ID NO:334); Kiss-1 whose activity liesmainly in its C-terminus(GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRF-NH2 (SEQ IDNO:335); EKDLPNYNWNSFGLRF-NH2 (SEQ ID NO:336)); RF-amide-3, whoseC-terminal fragments possess activity(SAGATANLPLRSGRNMEVSLVRRVPNLPQRF-NH2 (SEQ ID NO:337); VPNLPQRF-NH2 (SEQID NO:338)); Dynorphin, which exists as big dynorphin (A) of dynorphin B(rimorphin) (YGGFLRRIRPKLKWDNQKRYGGFLRRQFKVVT-OH (SEQ ID NO:339);YGGFLRRQFKVVT-OH (SEQ ID NO:340));

PYY whose C-terminal fragments are active at Y2 receptor(YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-NH2 (SEQ ID NO:57);SLRHYLNLVTRQRY-NH2 (SEQ ID NO:341)); AFP-6 whose 7-47 region retainsactivity (TQAQLLRVGCVLGTCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY-NH2 (SEQ IDNO:351); VGCVLGTCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY-NH2 (SEQ ID NO:52));the amylin family including adrenomdullin, calcitonin and CGRP; Oxytocinwhose C-terminal amide is generally needed for activity and can tolerateN-terminal extensions.

Exemplary peptide modules for use in the invention further include,C-Terminally extendable peptide modules including, Endothelin I, II andIII: ETI (CSCSSLMDKECVYFCHLDIIWVNTPEHVVPYGLGSPRS-OH (SEQ ID NO:342);CSCSSLMDKECVYFCHLDIIW-OH (SEQ ID NO:343)), ETII(CSCSSWLDKECVYFCHLDIIWVNTPEQTAPYGLGNPP-OH (SEQ ID NO:344);CSCSSWLDKECVYFCHLDIIW-OH (SEQ ID NO:345)) and ETIII(CTCFTYKDKECVYYCHLDIIWINTPEQTVPYGLSNYRGSFR-NH2 (SEQ ID NO:346);CTCFTYKDKECVYYCHLDIIW-OH (SEQ ID NO:347)); ghrelin whose activity liesmainly in its first 10 residues (GSSFLSPEHQRVQQRKESKKPPAKLQP-OH (SEQ IDNO:348); GSSFLSPEHQ-OH (SEQ ID NO:349)); glucagons, includingoxyntomodulin which is a C-terminally extended glucagon withglucagons-like activity (HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA-OH (SEQID NO:350); HSQGTFTSDYSKYLDSRRAQDFVQWLMNT-OH (SEQ ID NO:351));GLP-1/GLP-2 whose activities are retained with or without a C-terminalamide; GIP, which circulates in 2 forms, GIP1-42 and GIP1-30, both fullyactive at GIP Receptor (YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ-OH(SEQ ID NO:352); YAEGTFISDYSIAMDKIHQQDFVNWLLAQK-NH2 (SEQ ID NO:353));neuropeptide W, which exists as NPW23 and NPW30, equally active at GPR7and 8 (WYKHVASPRYHTVGRAAGLLMGLRRSPYLW-OH (SEQ ID NO:354);WYKHVASPRYHTVGRAAGLLMGL-OH (SEQ ID NO:355)); PACAP which exists in 2forms, PACAP27 and 38 (HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK-NH2 (SEQID NO:356); HSDGIFTDSYSRYRKQMAVKKYLAAVL-NH2 (SEQ ID NO:357)); PHI andPHV (HADGVFTSDFSKLLGQLSAKKYLESLMGKRVSSNISEDPVPV-OH (SEQ ID NO:358);HADGVFTSDFSKLLGQLSAKKYLESLM-NH2 (SEQ ID NO:359)); GRF, which exists in 2forms GRF29 and GRF40 (YADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARL-NH2(SEQ ID NO:360); YADAIFTNSYRKVLGQLSARKLLQDIMS-OH (SEQ ID NO:361)); PTH1-34 and 1-37 forms which possess activity of full length PTH 1-84(SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ (SEQ ID NO:362);SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVAL-OH (SEQ ID NO:363);SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF-OH (SEQ ID NO:364)); PTH-RP for which1-36 possesses activity of full length 1-86(AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAEIRATSEVSPNSKPSPNTKNHPVRFGSDDEGRYLTQETNKVETYKEQPLKTPGKKKKGKP-NH2 (SEQ ID NO:365);AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAEI-OH (SEQ ID NO:366)); gamma-MSH forwhich the shorter gamma-MSH1 and the longer gamma-MSH3 have similaractivities (YVMGHFRWDRFGRRNSSSSGSSGAGQ-OH (SEQ ID NO:367);YVMGHFRWDRF-NH2 (SEQ ID NO:368)); MSH for which alpha-MSH is an activeportion of ACTH (SYSMEHFRWGKPVGKKRRPVKVYPNGAEDESAEAFPLEF-OH (SEQ IDNO:369); SYSMEHFRWGKPV-NH2 (SEQ ID NO:370)); and endorphins for whichthe A, delta, and y endorphin are active subpeptides of the larger βendorphin (YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE-OH (SEQ ID NO:371);YGGFMTSEKSQTPLVTLFKNAIIKNAY-OH (SEQ ID NO:372); YGGFMTSEKSQTPLVTL-OH(SEQ ID NO:373); YGGFMTSEKSQTPLVT-OH (SEQ ID NO:374)).

The analogs of the above component peptide hormones are known in theart, but generally include modifications such as substitutions,deletions, and insertions to the amino acid sequence of such componentpeptide hormones, and any combination thereof. The substitutions,insertions and deletions may be at the N-terminal or C-terminal end, ormay be at internal portions of the component peptide hormone. In apreferred aspect, analogs of the component peptide hormones of theinvention include one or more modifications of a “non-essential” aminoacid residue. In the context of the invention, a “non-essential” aminoacid residue is a residue that can be altered, i.e., deleted orsubstituted, in the native human amino acid sequence of the fragment,e.g., the component peptide hormone fragment, without abolishing orsubstantially reducing the component peptide hormone receptor agonistactivity of the resulting analog.

Preferred substitutions include conserved amino acid substitutions. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain, or physicochemical characteristics (e.g., electrostatic, hydrogenbonding, isosteric, hydrophobic features). Families of amino acidresidues having similar side chains are known in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, methionine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,tryptophan), β-branched side chains (e.g., threonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine).

The present invention also relates to derivatives of the componentpeptide hormones. Such derivatives include component peptide hormonesand analogs thereof conjugated to one or more water soluble polymermolecules, such as polyethylene glycol (“PEG”) or fatty acid chains ofvarious lengths (e.g., stearyl, palmitoyl, octanoyl, etc.), or by theaddition of polyamino acids, such as poly-his, poly-arg, poly-lys, andpoly-ala. Modifications to the component peptide hormones or analogsthereof can also include small molecule substituents, such as shortalkyls and constrained alkyls (e.g., branched, cyclic, fused,adamantyl), and aromatic groups. The water soluble polymer moleculeswill preferably have a molecular weight ranging from about 500 to about20,000 Daltons.

Such polymer-conjugations and small molecule substituent modificationsmay occur singularly at the N- or C-terminus or at the side chains ofamino acid residues within the sequence of the hybrid polypeptides.Alternatively, there may be multiple sites of derivatization along thehybrid polypeptide. Substitution of one or more amino acids with lysine,aspartic acid, glutamic acid, or cysteine may provide additional sitesfor derivatization. See, e.g., U.S. Pat. Nos. 5,824,784 and 5,824,778.Preferably, the hybrid polypeptides may be conjugated to one, two, orthree polymer molecules.

The water soluble polymer molecules are preferably lined to an amino,carboxyl, or thiol group, and may be linked by N or C terminus, or atthe side chains of lysine, aspartic acid, glutamic acid, or cysteine.Alternatively, the water soluble polymer molecules may be linked withdiamine and dicarboxylic groups. In a preferred embodiment, the hybridpolypeptides of the invention are conjugated to one, two, or three PEGmolecules through an epsilon amino group on a lysine amino acid.

Derivatives of the invention also include component peptide hormones oranalogs with chemical alterations to one or more amino acid residues.Such chemical alterations include amidation, glycosylation, acylation,sulfation, phosphorylation, acetylation, and cyclization. The chemicalalterations may occur singularly at the N- or C-terminus or at the sidechains of amino acid residues within the sequence of the PPF hybridpolypeptides. In one embodiment, the C-terminus of these peptides mayhave a free —OH or —NH₂ group. In another embodiment, the N-terminal endmay be capped with an isobutyloxycarbonyl group, an isopropyloxycarbonylgroup, an n-butyloxycarbonyl group, an ethoxycarbonyl group, anisocaproyl group (isocap), an octanyl group, an octyl glycine group(G(Oct)), or an 8-aminooctanic acid group. In a preferred embodiment,cyclization can be through the formation of disulfide bridges.Alternatively, there may be multiple sites of chemical alteration alongthe hybrid polypeptide.

2. The Amylin Family

As discussed herein component peptide hormones useful in the presentinvention include amylin family peptide hormones including amylin,adrenomedullin (“ADM”), calcitonin (“CT”), calcitonin gene relatedpeptide (“CGRP”), intermedin (also known as “AFP-6”) and relatedpeptides. Native amylin family peptide hormones are known in art, as arefunctional peptide analogs and derivatives. Certain preferred nativepeptides, peptide analogs and derivatives are described herein, howeverit should be recognized that any known amylin family peptides thatexhibit hormonal activity known in the art may be used in conjunctionwith the present invention.

Any amylin analog or derivative known in the art may be used inconjunction with the present invention. In one embodiment, the amylinanalogs and derivatives have at least one hormonal activity of nativeamylin. In certain embodiments, the amylin analogs are agonists of areceptor which native amylin is capable of specifically binding.Preferred amylin analogs and derivatives include those described in US2003/0026812 A1, which is hereby incorporated by reference.

Exemplary amylin analogs include:

SEQ ID: 67 ^(25,28,29)Pro-h-amylin (pramlintide) 68 des-¹Lys-h-amylin 69²⁵Pro,²⁶Val,^(28,29)Pro-h-amylin 70 ¹⁸Arg,^(25,28)Pro-h-amylin 71des-¹Lys,¹⁸Arg,^(25,28)Pro-h-amylin 72 ¹⁸Arg,^(25,28,29)Pro-h-amylin 73des-¹Lys,¹⁸Arg,^(25,28,29)Pro-h-amylin 74des-¹,Lys^(25,28,29)Pro-h-amylin 75 ²⁵Pro,²⁶Val,^(28,29)Pro-h-amylin 76²⁸Pro-h-amylin, 2,7-Cyclo-[²Asp,⁷Lys]-h-amylin 77 ²⁻³⁷h-amylin 78¹Ala-h-amylin 79 ²Ala-h-amylin 80 ^(2,7)Ala-h-amylin 81 ¹Ser-h-amylin 82²⁹Pro-h-amylin 83 ^(25,28)Pro-h-amylin 84 des-¹Lys,^(25,28)Pro-h-amylin85 ²³Leu,²⁵Pro,²⁶Val,^(28,29)Pro-h-amylin 86²³Leu²⁵Pro²⁶Val²⁸Pro-h-amylin 87des-¹Lys,²³Leu,²⁵Pro,²⁶Val,²⁸Pro-h-amylin 88¹⁸Arg,²³Leu,²⁵Pro,²⁶Val,²⁸Pro-h-amylin 89¹⁸Arg,²³Leu,^(25,28,29)Pro-h-amylin 90 ¹⁸Arg²³Leu,^(25,28)Pro-h-amylin91 ¹⁷Ile,²³Leu,^(25,28,29)Pro-h-amylin 92 ¹⁷Ile,^(25,28,29)Pro-h-amylin93 des-¹Lys,¹⁷Ile,²³Leu,^(25,28,29)Pro-h-amylin 94¹⁷Ile,¹⁸Arg,²³Leu-h-amylin 95 ¹⁷Ile,¹⁸Arg,²³Leu,²⁶Val,²⁹Pro-h-amylin 96¹⁷Ile,¹⁸Arg,²³Leu,²⁵Pro,²⁶Val,^(28,29)Pro-h-amylin, 97¹³Thr,²¹His,²³Leu,²⁶Ala,²⁸Leu,²⁹Pro,³¹Asp-h-amylin 98¹³Thr,²¹His,²³Leu,²⁶Ala,²⁹Pro,³¹Asp-h-amylin 99des-¹Lys,¹³Thr,²¹His,²³Leu,²⁶Ala,²⁸Pro,³¹Asp-h-amylin 100¹³Thr,¹⁸Arg,²¹His,²³Leu,²⁶Ala,²⁹Pro,³¹Asp-h-amylin 101¹³Thr,¹⁸Arg,²¹His,²³Leu,^(28,29)Pro,³¹Asp-h-amylin 102¹³Thr,¹⁸Arg,²¹His,²³Leu,²⁵Pro,²⁶Ala,^(28,29)Pro,³¹Asp-h-amylin

As known in the art, such amylin analogs are preferably amidated, butwithin the context of the present invention, may optionally be in theacid form unless otherwise specified.

Any ADM analog or derivative known in the art may be used in conjunctionwith the present invention. In one embodiment, the ADM analogs andderivatives have at least one hormonal activity of native ADM. Incertain embodiments, the ADM analogs are agonists of a receptor whichnative ADM is capable of specifically binding.

Any CT analog or derivative known in the art may be used in conjunctionwith the present invention. In one embodiment, the CT analogs andderivatives have at least one hormonal activity of native CT. In certainembodiments, the CT analogs are agonists of a receptor which native CTis capable of specifically binding. Preferred CT analogs and derivativesinclude those described in U.S. Pat. Nos. 4,652,627; 4,606,856;4,604,238; 4,597,900; 4,537,716; 4,497,731; 4,495,097; 4,444,981;4,414,149; 4,401,593; and 4,397,780, which are hereby incorporated byreference.

Exemplary CT analogs include:

SEQ ID: 103 ⁸Gly-CT 104 ²²Leu-CT 105 ²Gly,³Ser,⁸Gly,²²des-Tyr-CT 106¹⁴Glu-sCT, 107 ¹⁸Arg-sCT, 108 ^(11,18)Arg-sCT, 109 ¹⁴Glu,¹⁸Arg-sCT, 110¹⁴Glu,^(11,18)Arg-sCT

As known in the art, such CT analogs are preferably amidated, but withinthe context of the present invention, may optionally be in the acid formunless otherwise specified.

Any CGRP analog or derivative known in the art may be used inconjunction with the present invention. In one embodiment, the CGRPanalogs and derivatives have at least one hormonal activity of nativeCGRP. In certain embodiments, the CGRP analogs are agonists of areceptor which native CGRP is capable of specifically binding. PreferredCGRP analogs and derivatives include those described in U.S. Pat. Nos.4,697,002; and 4,687,839, which are hereby incorporated by reference.

Exemplary CGRP analogs include:

SEQ ID: 111 ³⁶D-Ser-CGRP 112 ³⁶D-Thr-CGRP 113 ³⁶D-Asp-CGRP 114³⁶D-Asn-CGRP 115 ³⁶Ser-CGRP 116 ³⁶Hse-CGRP 117 ³⁶Asp-CGRP 118 ³⁶Thr-CGRP119 ³⁶Asn-CGRP

Any AFP-6 analog or derivative known in the art may be used inconjunction with the present invention. In one embodiment, the AFP-6analogs and derivatives have at least one hormonal activity of nativeAFP-6. In certain embodiments, the AFP-6 analogs are agonists of areceptor which native AFP-6 is capable of specifically binding.Preferred AFP-6 analogs and derivatives include those described in WO2003/022304, which is hereby incorporated by reference.

Exemplary AFP-6 analogs include:

SEQ ID: 120 TQAQLLRVGCGNLSTCQVQNLSHRLWQLMGPAGRQDSAPVD PSSPHSY 121TQAQLLRVGCDTATCQVQNLSHRLWQLMGPAGRQDSAPVDP SSPHSY 122TQAQLLRVGMVLGTMQVQNLSHRLWQLMGPAGRQDSAPVDP SSPHSY 123TQAQLLRVGCVLGTCQVQNLSHRLWQLMGPAGRQDSAPVEP SSPHSY 124TQAQLLRVGCVLGTCQVQNLSHRLWQLMGPAGRQESAPVEP SSPHSY 125TQAQLLRVGCVLGTCQVQNLSHRLWQL----RQDSAPVDPS SPHSY 126TQAQLLRVGCVLGTCQVQNLSHRLWQL----DSAPVDPSSP HSY 127RVGCVLGTCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY 128VGCVLGTCQVQNLSHRLWQLMGPAGRQDSAPVEPSSPHSY 129VGCVLGTCQVQNLSHRLWQL----RQDSAPVEPSSPHSY 130GCVLGTCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY 131GCNTATCQVQNLSHRLWQL----RQDSAPVDPSSPHSY 132GCNTATCQVQNLSHRLWQL----RQDSAPVEPSSPHSY 133GCSNLSTCQVQNLSHRLWQL----RQDSAPVEPSSPHSY 134GCGNLSTCQVQNLSHRLWQL----RQDSAPVEPSSPHSY 135GCVLGTCQVQNLSHRLWQL----RQESAPVEPSSPHSY 136CVLGTCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY 137QVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY 138 VQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY 139VQNLSHRL----QLMGPAGRQDSAPVDPSSPHSY 140GTMQVQNLSHRLWQL----RQDSAPVEPSSPHSY

As known in the art, such AFP-6 analogs are preferably amidated, butwithin the context of the present invention, may optionally be in theacid form unless otherwise specified.

3. The CCK Family

CCKs, including hCCK and species variants, and various analogs thereofare known in the art. Generally, CCK has a 33-amino acid sequence firstidentified in humans, and includes a 8-amino acid in vivo C-terminalfragment (“CCK-8”) that has been reportedly demonstrated in pig, rat,chicken, chinchilla, dog and humans. Other species variants include a39-amino acid sequence found in pig, dog and guinea pig, and a 58-aminoacid found in cat, dog and humans, and a 47-amino acid sequenceshomologous to both CCK and gastrin. The C-terminal tyrosine-sulfatedoctapeptide sequence (CCK-8) is relatively conserved across species, andmay be the minimum sequence for biological activity in the periphery ofrodents. Thus, the term CCK-33 will generally refer to human CCK(1-33),while CCK-8 (CCK(26-33); SEQ ID NO:55) will refer to the C-terminaloctapeptide generically in both the sulfated and unsulfated unlessotherwise specified. Further, pentagastrin or CCK-5 will refer to theC-terminal peptide CCK(29-33) (SEQ ID NO:209), and the CCK-4 will referto the C-terminal tetrapeptide CCK(30-33) (SEQ ID NO:208).

The type A receptor subtype (CCK_(A)) has been reported to be selectivefor the sulfated octapeptide. The Type B receptor subtype (CCK_(B)) hasbeen identified throughout the brain and in the stomach, and reportedlydoes not require sulfation or all eight amino acids.

Various in vivo and in vitro screening methods for CCK analogs are knownin the art. Examples include in vivo assays involving the contraction ofthe dog or guinea pig gallbladder after rapid intravenous injection ofthe compound to be tested for CCK-like activity, and in vitro assaysusing strips of rabbit gallbladder. See Walsh, “GastrointestinalHormones”, In Physiology of the Gastrointestinal Tract (3d ed. 1994;Raven Press, New York).

Certain exemplary CCKs and CCK analogs with CCK activity include:

SEQ ID: 141 DY(SO₃H)MGWMDF 142 DYMGWMDF 143 MGWMDF 144 GWMDF 145 WMDF146 KDY(SO₃H)MGWMDF 147 KDYMGWMDF 148 KMGWMDF 149 KGWMDF 150 KWMDF

As known in the art, such CCK peptides are preferably amidated, butwithin the context of the present invention, may optionally be in theacid form unless otherwise specified.

4. The Leptin Family

Component peptide hormones useful in the present invention also includeleptin family peptide hormones. Native leptin family peptide hormonesare known in art, as are functional peptide analogs and derivatives.Certain preferred native peptides, peptide analogs and derivatives aredescribed herein, however it should be recognized that any known amylinfamily peptides that exhibit hormonal activity known in the art may beused in conjunction with the present invention.

Any leptin analog or derivative known in the art may be used inconjunction with the present invention. In one embodiment, the leptinanalogs and derivatives have at least one hormonal activity of nativeleptin. In certain embodiments, the leptin analogs are agonists of areceptor which native leptin is capable of specifically binding.Preferred leptin analogs and derivatives include those described in,e.g., WO 2004/039832, WO 98/55139, WO 98/12224, and WO 97/02004, all ofwhich are hereby incorporated by reference.

Exemplary leptin analogs include those where the amino acid at position43 is substituted with Asp or Glu; position 48 is substituted Ala;position 49 is substituted with Glu, or absent; position 75 issubstituted with Ala; position 89 is substituted with Leu; position 93is substituted with Asp or Glu; position 98 is substituted with Ala;position 117 is substituted with Ser, position 139 is substituted withLeu, position 167 is substituted with Ser, and any combination thereof.

Certain exemplary leptin and leptin analogs with leptin activityinclude:

SEQ ID: 151 ⁴³Asp-leptin 152 ⁴³Glu-leptin 153 ⁴⁸Ala-leptin 154⁴⁹Glu-leptin 155 ⁴⁹Des-AA-leptin 156 ⁷⁵Ala-leptin 157 ⁸⁹Leu-leptin 158⁹³Asp-leptin 159 ⁹³Glu-leptin 160 ⁹⁸Ala-leptin 161 ¹¹⁷Ser-leptin 162¹³⁹Leu-leptin 163 ¹⁶⁷Ser-leptin 164 ⁴³Asp,⁴⁹Glu-leptin 165⁴³Asp,⁷⁵Ala-leptin 166 ⁸⁹Leu,¹¹⁷Ser-leptin 167 ⁹³Glu,¹⁶⁷Ser-leptin

5. The PPF Family

Component peptide hormones useful in the present invention also includePPF peptide hormones, including PP and PYY. Native PPF peptide hormonesare known in art, as are functional peptide analogs and derivatives.Certain preferred native peptides, peptide analogs and derivatives aredescribed herein, however it should be recognized that any known amylinfamily peptides that exhibit hormonal activity known in the art may beused in conjunction with the present invention.

Any PPF analog or derivative known in the art may be used in conjunctionwith the present invention. In one embodiment, the PPF analogs andderivatives have at least one hormonal activity of a native PPFpolypeptide. In certain embodiments, the PPF analogs are agonists of areceptor which native PPF polypeptide is capable of specificallybinding. Preferred PPF analogs and derivatives include those describedin WO 03/026591 and WO 03/057235, which are herein incorporated byreference in their entirety.

In one embodiment, preferred PPF analogs and derivatives that exhibit atleast one PPF hormonal activity generally comprise at least two PYYmotifs including a polyproline motif and C-terminal tail motif. Suchanalogs are generally described in U.S. Provisional Application No.60/543,406 filed Feb. 11, 2004, which is herein incorporated byreference. Other preferred PPF analogs are disclosed inPCT/US2005/004351, entitled “Pancreatic Polypeptide Family Motifs andPolypeptides Comprising the Same”, Attorney Docket 18528.832, thecontents of which is hereby incorporated by reference. By way ofbackground, research has suggested that the differences in Y receptorbinding affinities are correlated with secondary and tertiary structuraldifferences. See, e.g., Keire et al., Biochemistry 2000, 39, 9935-9942.Native porcine PYY has been characterized as including two C-terminalhelical segments from residues 17 to 22 and 25 to 33 separated by a kinkat residues 23, 24, and 25, a turn centered around residues 12-14, andthe N-terminus folded near residues 30 and 31. Further, full-lengthporcine PYY has been characterized as including the PP fold, stabilizedby hydrophobic interactions among residues in the N- and C-termini. Seeid.

A “PYY motif” is generally a structural component, primary, secondary,or tertiary, of a native PP family polypeptide that is critical tobiological activity, i.e., biological activity is substantiallydecreased in the absence or disturbance of the motif. Preferred PYYmotifs include the N-terminal polyproline type II motif of a native PPfamily polypeptide, the type II β-turn motif of native PP familypolypeptide, the α-helical motif at the C-terminal end of native PPfamily polypeptide, and the C-terminal tail motif of native PP familypolypeptide. More particularly, in the N-terminal polyproline region,amino acids corresponding to residues 5 and 8 of a native PP familypolypeptide are generally conserved as a proline. The type II β-turnmotif will generally include amino acids corresponding to residues 12-14of a native PP family polypeptide. The α-helical motif can generallyextend from amino acids corresponding to approximately residue 14 of anative PP family polypeptide to any point up to and including theC-terminal end, so long as the α-helical motif includes a sufficientnumber of amino acid residues such that an α-helical turn is formed insolution. The α-helical motif can also include amino acid substitutions,insertions and deletions to the native PP family sequence, so long asthe α-helical turn is still formed in solution. The C-terminal tailmotif generally includes amino acids corresponding to approximately thelast 10 residues of a native PP family polypeptide, more preferably thelast 7, 6, or 5 residues of a native PP family polypeptide, and morepreferably amino acid residues 32-35.

Preferred PYY analogs include those with internal deletions, insertions,and substitutions in areas of the PYY molecule not corresponding to thepolyproline motif and/or the C-terminal tail motif. For instance,internal deletions at positions 4, 6, 7, 9, or 10 are envisioned.

6. Incretins and Incretin Mimetics

Component peptide hormones useful in the present invention also includeGLP-1 peptide hormones. Native GLP-1 peptide hormones, includingGLP-1(1-37) (SEQ ID NO:59), GLP-1(7-37) (SEQ ID NO:204), andGLP-1(7-36)amide (SEQ ID NO:61), are known in art, as are functionalpeptide analogs and derivatives. As used herein, GLP-1 refers to allnative forms of GLP-1 peptide hormones. Certain preferred nativepeptides, peptide analogs and derivatives are described herein, howeverit should be recognized that any known GLP-1 peptides that exhibithormonal activity known in the art may be used in conjunction with thepresent invention.

Any GLP-1 peptide analog or derivative known in the art may be used inconjunction with the present invention. In one embodiment, the GLP-1peptide analogs and derivatives have at least one hormonal activity of anative GLP-1 peptide. In certain embodiments, the GLP-1 peptide analogsare agonists of a receptor which a native GLP-1 peptide is capable ofspecifically binding. Preferred GLP-1 peptide analogs and derivativesinclude those described in, e.g., WO 91/11457, which is herebyincorporated by reference.

GLP-1 analogs known in the art include:

SEQ ID NO: 168 ⁹Gln-GLP-1(7-37) 169 D-⁹Gln-GLP-1(7-37) 170¹⁶Thr-¹⁸Lys⁻GLP-1(7-37) 171 ¹⁸Lys-GLP-1(7-37) 172 ⁸Gly-GLP-1 (7-36) 173⁹Gln-GLP-1 (7-37) 174 D-⁹Gln-GLP-1 (7-37) 175 acetyl-⁹Lys-GLP-1(7-37)176 ⁹Thr-GLP-1(7-37) 177 D-⁹Thr-GLP-1 (7-37) 178 ⁹Asn-GLP-1 (7-37) 179D-⁹Asn-GLP-1 (7-37) 180 ²²Ser²³Arg²⁴Arg²⁶Gln-GLP-1(7-37) 181¹⁶Thr¹⁸Lys-GLP-1(7-37) 182 ¹⁸Lys-GLP-1(7-37) 183 ²³Arg-GLP-1(7-37) 184²⁴Arg-GLP-1(7-37)

As known in the art, such GLP-1 analogs may preferably be amidated, butwithin the context of the present invention, may optionally be in theacid form unless otherwise specified.

Other GLP-1 analogs and derivatives are disclosed in U.S. Pat. No.5,545,618 which is incorporated herein by reference. A preferred groupof GLP-1 analogs and derivatives include those disclosed in U.S. Pat.No. 6,747,006, which is herein incorporated by reference in itsentirety. The use in the present invention of a molecule described inU.S. Pat. No. 5,188,666, which is expressly incorporated by reference,is also contemplated. Another group of molecules for use in the presentinvention includes compounds described in U.S. Pat. No. 5,512,549, whichis expressly incorporated herein by reference. Another preferred groupof GLP-1 compounds for use in the present invention is disclosed in WO91/11457, which is herein incorporated by reference.

Component peptide hormones useful in the present invention also includeGLP-2 peptide hormones. Native GLP-2 peptide hormones, e.g., rat GLP-2and its homologous including ox GLP-2, porcine GLP-2, degu GLP-2, bovineGLP-2, guinea pig GLP-2, hamster GLP-2, human GLP-2, rainbow troutGLP-2, and chicken GLP-2, are known in art, as are functional peptideanalogs and derivatives. Certain preferred native peptides, peptideanalogs and derivatives are described herein, however it should berecognized that any known GLP-2 peptides that exhibit hormonal activityknown in the art may be used in conjunction with the present invention.

Any GLP-2 peptide analog or derivative known in the art may be used inconjunction with the present invention. In one embodiment, the GLP-2peptide analogs and derivatives have at least one hormonal activity of anative GLP-2 peptide. In certain embodiments, the GLP-2 peptide analogsare agonists of a receptor which a native GLP-2 peptide is capable ofspecifically binding. Preferred GLP-2 peptide analogs and derivativesinclude those described in, e.g., U.S. Ser. No. 08/669,791 and PCTApplication PCT/CA97/00252, both of which are hereby incorporated byreference. Specific GLP-2 analogs known in the art include: rat or humanGLP-2 altered at position 2 to confer DPP-IV resistance by substitutinga Gly for an Ala.

Component peptide hormones useful in the present invention also includeoxyntomodulin (OXM) peptide hormones. Native OXM peptide hormones areknown in art, as are functional peptide analogs and derivatives. Certainpreferred native peptides, peptide analogs and derivatives are describedherein, however it should be recognized that any known OXM peptides thatexhibit hormonal activity known in the art may be used in conjunctionwith the present invention.

Any OXM peptide analog or derivative known in the art may be used inconjunction with the present invention. In one embodiment, the OXMpeptide analogs and derivatives have at least one hormonal activity of anative OXM peptide. In certain embodiments, the OXM peptide analogs areagonists of a receptor which a native OXM peptide is capable ofspecifically binding.

Component peptide hormones useful in the present invention also includeexendin peptide hormones. Native exendin peptide hormones are known inart, as are functional peptide analogs and derivatives. Certainpreferred native peptides, peptide analogs and derivatives are describedherein, however it should be recognized that any known exendin peptidesthat exhibit hormonal activity known in the art may be used inconjunction with the present invention.

Any exendin peptide analog or derivative known in the art may be used inconjunction with the present invention. In one embodiment, the exendinpeptide analogs and derivatives have at least one hormonal activity of anative exendin peptide. In certain embodiments, the exendin peptideanalogs are agonists of a receptor which a native exendin peptide iscapable of specifically binding.

Exemplary exendin analogs include:

SEQ ID NO: 185 ¹⁴Leu,²⁵Phe-exendin-4 186 ⁵Ala,¹⁴Leu,²⁵Phe-exendin-4 187¹⁴Leu,²²Ala,²⁵Phe-exendin-4

As known in the art, such exendin analogs are preferably amidated, butwithin the context of the present invention, may optionally be in theacid form unless otherwise specified.

Additional exemplary exendin analogs and derivatives are described inPCT Application Serial No. PCT/US98/16387 filed Aug. 6, 1998, entitled“Novel Exendin Agonist Compounds,” which claims the benefit of U.S.patent application Ser. No. 60/055,404, filed Aug. 8, 1997, both ofwhich are herein incorporated by reference. Other exendin analogs andderivatives are described in PCT Application Serial No. PCT/US98/24210,filed Nov. 13, 1998, entitled “Novel Exendin Agonist Compounds,” whichclaims the benefit of U.S. Provisional Application No. 60/065,442 filedNov. 14, 1997, both of which are herein incorporated by reference. Stillother exendin analogs and derivatives are described in PCT ApplicationSerial No. PCT/US98/24273, filed Nov. 13, 1998, entitled “Novel ExendinAgonist Compounds,” which claims the benefit of U.S. ProvisionalApplication No. 60/066,029 filed Nov. 14, 1997, both of which are hereinincorporated by reference. Still other exendin analogs and derivativesare described in PCT Application Serial No. PCT/US97/14199, filed Aug.8, 1997, entitled “Methods for Regulating Gastrointestinal Activity,”which is a continuation-in-part of U.S. patent application Ser. No.08/694,954 filed Aug. 8, 1996, both of which are hereby incorporated byreference. Still other exendin analogs and derivatives are described inPCT Application Serial No. PCT/US98/00449, filed Jan. 7, 1998, entitled“Use of Exendins and Agonists Thereof for the Reduction of Food Intake,”which claims priority to U.S. Provisional Application No. 60/034,905filed Jan. 7, 1997, both of which are hereby incorporated by reference.Yet other exendin analogs and derivatives are described in US2004/0209803 A1, filed Dec. 19, 2003, entitled “Compositions for theTreatment and Prevention of Neuropathy,” which is hereby incorporated byreference.

7. Natriuretic Peptides.

Natriuretic peptides are a family of hormones that consist of atrialnatriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-typenatriuretic peptide (CNP). They are synthesized and stored as 3 distinctprecursor prohormones, which are the 126 amino acid ANP, 108 amino acidBNP, and 104 amino acid CNP. They are each encoded by separate genes andhave distinct sites of synthesis and mechanisms of regulation. Parentalnatriuretic peptide sequences include:

SEQ ID 151 amino acid MSSFSTTTVSFLLLLAFQLLGQTRANPMYNAVSNADL NO: 375human ANP MDFKNLLDHLEEKMPLEDEVVPPQVLSDPNEEAGAA preprohormoneLSPLPEVPPWTGEVSPAQRDGGALGRGPWDSSDRSALLKSKLRALLTAPRSLRRSSCFGGRMDRIGAQSGLGCN SFRY SEQ ID 134 amino acidMDPQTAPSRALLLLLFLHLAFLGGRSHPLGSPGSASDL NO: 376 human BNPETSGLQEQRNHLQGKLSELQVEQTSLEPLQESPRPTGV preprohormoneWKSREVATEGIRGHRKMVLYTLRAPRSPKMVQGSGC FGRKMDRISSSSGLGCKVLRRH SEQ ID126 amino acid MHLSQLLACALLLTLLSLRPSEAKPGAPPKVPRTPPAE NO: 377 human CNPELAEPQAAGGGQKKGDKAPGGGGANLKGDRSRLLR preproCNPDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSK GCFGLKLDRIGSMSGLGC

The main site of synthesis of the ANP prohormone is the atrial myocytewhere it is synthesized as a 151-amino acid preprohormone. Removal of a25-amino acid signal peptide from its N terminal end occurs in theendoplasmic reticulum, leaving a 126-amino acid ANP prohormone (ProANP),the main storage form of ANP within the heart. The prohormone consistsof 4 biologically active peptide segments: amino acids 1-30 (ProANF1-30, also known as long acting Na stimulator), 31-67 (ProANF 31-67,also known as vessel dilator), 79-98 (ProANF 79-98, also known aspotassium excreter), and 99-126 (ANF, also known as atrial natriureticfactor).

BNP was originally isolated from porcine brain but in humans it issynthesized and secreted from the left ventricle. Sequence analysisreveals that preproBNP consists of 134 residues and is cleaved to a108-amino acid ProBNP. Cleavage of a 32-amino acid sequence from theC-terminal end of ProBNP results in human BNP (77-108), which is thephysiologically active form in plasma.

CNP is the third member of the natriuretic peptide system and isprimarily found in human vascular endothelial cells, kidney, and porcinebrain. High concentrations of CNP are also found in human hypothalamusand midbrain. In humans, preproCNP is a 126-amino acid precursorprocessed into proCNP by cleavage of 23 residues from its N-terminalend. This 23-amino acid sequence serves as a signal peptide. Theterminal 22 (105-126) amino acids are cleaved from proCNP to yield abiologically active form of CNP.

Urodilatin is a kidney-derived member of the natriuretic peptide familyand is formed from the same ANP prohormone and consists of amino acids95-126. Except for the 4 amino acid N terminal extension, it isidentical to ANF (99-126). Urodilatin appears to be an importantregulator of sodium and water handling in the kidney, as well as amediator of sodium excretion in patients with congestive heart failure(CHF).

Natriuretic peptides exert their biologic effects by binding tohigh-affinity receptors on the surface of target cells. Three subtypesof NPRs—NPR-A, NPR-B, and NPRC—have been isolated. Consequently, in oneembodiment is provided a method to screen hybrids for natriureticreceptor binding and/or activation. Natriuretic peptides includingprohormone variants can impart numerous natriuretic peptide hormoneactivities to hybrids of the invention. In other embodiments of interestare natriuretic antagonist hybrids. Natriuresis is the excretion of anexcessively large amount of sodium into the urine. Natriuresis issimilar to diuresis (the excretion of an unusually large quantity ofurine), except that in natriuresis the urine is exceptionally salty.Natriuresis occurs with some diuretics and diseases (as of the adrenal)and can lead to the salt-losing syndrome characterized by dehydration,vomiting, low blood pressure, and the risk of sudden death. Exogenousadministration of the 4 independent circulating peptides of the ANPprohormone (1-30, 31-67, 79-98, and 99-126) produce in vivovasodilation, diuresis, suppression of the renin-angiotensin-aldosteronesystem and enhanced natriuresis and/or kaliuresis. ProANF 1-30, ProANF31-67 and ANF 99-126 each have natriuretic, blood pressure lowering anddiuretic properties with ProANF 31-67 and ANF 99-126 having the greatestimpact on blood pressure. There are varying effects of the ANP peptideson potassium homeostasis: ProANF 79-98 stimulates potassium excretion,whereas ProANF 31-67 spares potassium loss by inhibiting Na/K ATPase inthe medullary collecting duct cells. Specific to ANF 99-126 is adose-dependent inhibition of angiotensin II-mediated aldosteronesecretion, whereas proANF 31-67 has the property of inducing natriuresisthrough generation of prostaglandin.

BNP produces similar biologic effects as ANF in normal humans. Infusionsof BNP in normal men produced a 2-fold increase in sodium excretion, 50%reduction in plasma renin, angiotensin II and aldosterone secretion aswell as a reduction in plasma volume.

CNP induces cardiovascular effects similar to the other natriureticpeptides but does not appear to mediate any renal effects. When CNP isinfused in anesthetized dogs at equivalent doses of ANF, plasma cGMProse with a concomitant reduction in mean arterial pressure, rightatrial pressure and cardiac output, but glomerular filtration rate,renal blood flow and sodium excretion decreased.

Natriuretic peptides can provide therapeutic benefit in heart failure.Congestive heart failure (CHF) is associated with increases invasopressin, endothelin, and with activation of therenin-angiotensin-aldosterone system, and sympathetic nervous systems,mediating vasoconstriction, sodium and water retention, and negativevascular and cardiac remodeling. These effects occur despite theelevated levels of the natriuretic peptides in patients with heartfailure. In one embodiment of the invention are hybrids that provideincreased or therapeutic serum levels of natriuretic peptide activityfor treatment or prevention of cardiac related diseases and conditions,including CHF. Although ANF infusion in normal individuals can result ina sustained increase in sodium excretion and urine flow rates, in theheart failure patient a marked beneficial reduction in renal responsecan be obtained. BNP infusion markedly increases sodium excretion inpatients with heart failure and exerts significant beneficialhemodynamic effects. As compared with ANP, the diuretic and natriureticeffects of BNP are significantly greater. BNP is cleared more slowlythan ANP and exerts other effects including suppressing aldosteronesecretion and increasing serum levels of ANP. BNP peptides can alsoprovide a beneficial decrease in pulmonary capillary wedge pressure,systemic vascular resistance, right atrial pressure and systolic bloodpressure, with an increase in cardiac index in patients hospitalized forsymptomatic CHF. In patients with decompensated heart failure,natriuretic peptide hybrids can provide a beneficial decrease inpulmonary capillary wedge pressure and an improved dyspnea score.(Dyspnea is an unpleasant sensation of difficulty in breathing,typically associated with early stages of cardiac heart failure.) Thehybrids containing one, two or three natriuretic hormone functionsprovide methods of administration of pharmaceutically activecompositions that are useful for both the prophylactic and therapeutictreatment of CHF patients, preferably CHF patients that aredecompensated, patients with chronic CHF, and patients withhypertension. The natriuretic portion(s) of a hybrid is sufficient toprovide a therapeutically effective amount of a natriurertic peptide tosuch patient when administered in a therapeutically effective dose overa therapeutically effective period.

As discussed herein any of the family of therapeutically effectivenatriuretic peptides or their analogs can be used. Useful natriureticpeptides include, for example, atrial natriuretic peptide (ANP), brainnatriuretic peptide (BNP or B-type natriuretic peptide) and C-typenatriuretic peptide (CNP). Sequences of useful forms of natriureticpeptides are disclosed in U.S. Patent Publication 20010027181, which isincorporated herein by reference. Examples of ANPs include human ANP(Kangawa et al., BBRC 118:131 (1984)) or that from various species,including pig and rat ANP (Kangawa et al., BBRC 121:585 (1984)). SuchANPs comprise 28 amino acids. Such ANPs may be administered as a peptidehaving a ring structure of ANP (formation of a disulfide bond based onCys), and a C-terminal portion succeeding the ring structure. An exampleof such a peptide is a peptide having amino acid residues at the7-position to the 28-position of ANP is provided in U.S. PatentApplication Publication No. 20010027181. Another example is frog ANP.Specific examples of BNPs that can be used in the methods of theinvention include human BNP (hBNP). Human BNP comprises 32 amino acidsand involves the formation of a disulfide bond (Sudoh et al., BBRC159:1420 (1989)) and U.S. Pat. Nos. 5,114,923, 5,674,710, 5,674,710, and5,948,761, each of which is incorporated by reference. Various BNP's oforigin other than human, including as pig BNP and rat BNP, are alsoknown, and can be used. A further example is chicken BNP. Examples ofCNPs that can be used in the methods of the invention include pig CNP.Pig CNP comprises 22 amino acids and involves the formation of adisulfide bond, like the above-described ANP and BNP (Sudoh et al., BBRC168:863 (1990)) (human and rat have the same amino acid sequence),chicken CNP (Arimura et al., BBRC 174:142 (1991)). Frog CNP (Yoshiharaet al., BBRC 173:591 (1990) can also be used. As discussed herein, oneskilled in the art can apply modifications, such as a deletion,substitution, addition or insertion, and/or chemical modification toamino acid residues in the amino acid sequence of a known natriureticpeptide as desired, by known methods. The resulting compound is acompound which has the activity of acting on a receptor of the startingANP, BNP or CNP. Analogs having this activity, therefore, are includedin the hybrids for use in accordance with the methods of the presentinvention.

In another embodiment, the hybrids containing one or more natriureticfunctions can be used in treating hypertension. In one embodiment anatriuretic hybrid will have no deleterious effect on heart rate and isnot associated with arrhythmias. In one embodiment the hybrid willcomprise at least one, two or three natriuretic peptide functions, forexample, both ANP and BNP activity. One or more natriuretic hormonefunctions can be combined with any other hormone function or peptidicenhancer, as described herein. In another embodiment the natriureticportion(s) is a more stable analog having an extended in vivo half-lifewhen compared with that of a native natriuretic peptide. Analogs thatprevent undesirable cleavage by endogenous enzymes such as NEP are alsoenvisioned. The natriuretic containing hybrids are also further directedto hypertension reduction, diuresis inducement, natriuresis inducement,vascular conduct dilatation or relaxation, natriuretic peptide receptors(such as NPR-A) binding, renin secretion suppression from the kidney,aldostrerone secretion suppression from the adrenal gland, treatment ofcardiovascular diseases and disorders, reducing, stopping or reversingcardiac remodeling in congestive heart failure, treatment of renaldiseases and disorders; treatment or prevention of ischemic stroke, andtreatment of asthma. Hybrids can be administered to patients that wouldbenefit from inducing natriuresis, diuresis and vasodilatation. Hybridscan be administered alone or in combination with one or more of thefollowing types of compounds: ACE inhibitors, beta-blockers, diuretics,spironolactone, digoxin, anticoagulation and antiplatelet agents, andangiotensin receptor blockers. Additional diseases or conditions includerenal disorders and diseases, asthma, hypertension and pulmonaryhypertension. Hybrids are also useful to treat inflammatory-relateddiseases, erectile dysfunction and hypercholesterolemia.

Bio-Active Peptide Hormone Modules

As discussed herein the hybrid polypeptides of the present inventiongenerally comprise at least two bio-active peptide hormone modulescovalently linked together. The bio-active peptide hormone modules maybe: (a) native component peptide hormones, (b) analogs or derivatives ofnative component peptide hormones that retain hormonal activity, (c)fragments of native component peptide hormones that retain hormonalactivity, (d) fragments of analogs or derivatives of native componentpeptide hormones that retain hormonal activity, (e) structural motifs ofnative component peptide hormones that impart a desired chemicalstability, conformational stability, metabolic stability,bioavailability, organ/tissue targeting, receptor interaction, proteaseinhibition, plasma protein binding, and/or other pharmacokineticcharacteristic to the hybrid polypeptide; or (f) structural motifs ofanalogs or derivatives of native component peptide hormones that imparta desired chemical stability, conformational stability, metabolicstability, bioavailability, organ/tissue targeting, receptorinteraction, protease inhibition, plasma protein binding, and/or otherpharmacokinetic characteristic to the hybrid polypeptide. The structuralmotifs of (e) and (f) will collectively be referred to herein as“peptidic enhancers”.

Preferred bio-active peptide hormone modules include native peptidehormones selected from: amylin, ADM, CT, CGRP, intermedin, CCK(1-33),CCK-8, leptin, PYY(1-36) (SEQ ID NO:57), PYY(3-36) (SEQ ID NO:58),GLP-1(1-37) (SEQ ID NO:59), GLP-1(7-37) (SEQ ID NO:204), GLP-1(7-36)(SEQ ID NO:61), GLP-2, OXM, exendin-3, exendin-4, natriuretic peptidehormones, urocortin family peptides, e.g., Ucn-2 and Ucn-3, neuromedinfamily peptides, e.g. neuromedin U25 or splice variants, and ANP, BNP,CNP or urodilatin.

Other preferred bio-active peptide hormone modules include analogs andderivatives of a component peptide hormone selected from: amylin, ADM,CT, CGRP, intermedin, CCK, leptin, PYY(1-36) (SEQ ID NO:57), PYY(3-36)(SEQ ID NO:58), GLP-1(1-37) (SEQ ID NO:59), GLP-1(7-37) (SEQ ID NO:204),GLP-1(7-36) (SEQ ID NO:61), GLP-2, OXM, exendin-3, and exendin-4,natriuretic peptide hormones, urocortin family peptides, e.g., Ucn-2 andUcn-3, neuromedin family peptides, e.g. neuromedin U25 or splicevariants, and ANP, BNP, CNP or urodilatin, wherein the analog orderivative exhibits at least one hormonal activity of the componentpeptide hormone. The analog may comprise one or more insertions,deletions, or substitutions of the amino acid sequence of the componentpeptide hormone, and the derivative may comprise one or more chemicalmodifications of an amino acid residue of an analog or component peptidehormone, as described more fully herein and known in the art.

More specifically, analogs and derivatives may be selected from anydescribed above and/or known in the art. Particularly preferred analogsand derivatives that exhibit at least one hormonal activity useful asbio-active peptide hormone modules of the invention include thefollowing:

Amylin:²Ala-h-amylin (SEQ ID NO: 79), ^(2,7)Ala-h-amylin (SEQ ID NO: 80), ²⁸Pro-h-amylin (SEQ ID NO: 189), ^(25,28)Pro-h-amylin (SEQ ID NO: 83),^(25,28,29)Pro-h-amylin (SEQ ID NO: 67), ²⁵Pro,²⁶Va1,^(28,29)Pro-h-amylin (SEQID NO: 69), ¹⁸Arg,^(25,28)Pro-h-amylin (SEQ ID NO: 70), ¹⁸Arg,^(25,28,29)Pro-h-amylin (SEQ ID NO: 72), ²⁵Pro,²⁶Va1,^(28,29)Pro-h-amylin (SEQ ID NO: 75),¹⁸Arg,²³Leu,^(25,28,29)Pro-h-amylin (SEQ ID NO: 89), ¹⁸Arg²³Leu,^(25,28)Pro-h-amylin (SEQ ID NO: 90), and 2,7-Cyclo-[²Asp,⁷Lys]-h-amylin (SEQ IDNO: 76) CT:¹⁴Glu-sCT (SEQ ID NO: 106), ¹⁸Arg-sCT (SEQ ID NO: 107), ^(11,18)Arg-sCT(SEQ ID NO: 108), ¹⁴Glu,¹⁸Arg-sCT (SEQ ID NO: 109), ¹⁴Glu,^(11,18)Arg-sCT (SEQ ID NO: 110) CGRP:³⁶D-Ser-CGRP (SEQ ID NO: 111), ³⁶D-Thr-CGRP (SEQ ID NO: 112),³⁶D-Asp-CGRP (SEQ ID NO: 113), ³⁶D-Asn-CGRP (SEQ ID NO: 114),³⁶Ser-CGRP (SEQ ID NO: 115), ³⁶Hse-CGRP (SEQ ID NO: 116), ³⁶Asp-CGRP (SEQ ID NO: 117), ³⁶Thr-CGRP (SEQ ID NO: 118), ³⁶Asn-CGRP(SEQ ID NO: 119) AFP-6: TQAQLLRVGCGNLSTCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY (SEQ ID NO: 120), TQAQLLRVGCDTATCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY (SEQ ID NO: 121), TQAQLLRVGMVLGTMQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY (SEQ ID NO: 122), TQAQLLRVGCVLGTCQVQNLSHRLWQLMGPAGRQDSAPVEPSSPHSY (SEQ ID NO: 123), TQAQLLRVGCVLGTCQVQNLSHRLWQLMGPAGRQESAPVEPSSPHSY (SEQ ID NO: 124), CCK:DY(OSO₃H)MGWMDF (SEQ ID NO: 141), DYMGWMDF (SEQ IDNO: 142), MGWMDF (SEQ ID NO: 143), GWMDF (SEQ ID NO: 144),WMDF (SEQ ID NO: 145), KDY(OSO₃H)MGWMDF (SEQ ID NO: 146),KDYMGWMDF (SEQ ID NO: 147), KMGWMDF (SEQ ID NO: 148),KGWMDF (SEQ ID NO: 149), KWMDF (SEQ ID NO: 150) Leptin:⁴³Asp-leptin (SEQ ID NO: 151), ⁴³Glu-leptin (SEQ ID NO: 152), ⁴⁸Ala-leptin (SEQ ID NO: 153), ⁴⁹Glu-leptin (SEQ ID NO: 154), ⁴⁹Des-AA-leptin (SEQ ID NO: 155), ⁷⁵Ala-leptin (SEQ ID NO: 156), ⁸⁹Leu-leptin(SEQ ID NO: 157), ⁹³Asp-leptin (SEQ ID NO: 158), ⁹³Glu-leptin (SEQ IDNO: 159), ⁹⁸Ala-leptin (SEQ ID NO: 160), ¹³⁹Leu-leptin (SEQ ID NO: 162),PYY: ³Leu-PYY (SEQ ID NO: 192), ³Val-PYY (SEQ ID NO: 193), ⁴Arg-PYY(SEQ ID NO: 194), ⁴Gln-PYY (SEQ ID NO: 195), ⁴Asn-PYY (SEQ IDNO: 196), ²⁵Lys-PYY (SEQ ID NO: 197), ³⁴Pro-PYY (SEQ ID NO: 198),³⁴His-PYY (SEQ ID NO: 199), ^(1,36)Tyr-PYY (SEQ ID NO: 57), ¹³Pro¹⁴Ala-PYY (SEQ ID NO: 200), ³¹Leu³⁴Pro-PYY (SEQ ID NO: 201), des-AA-4-PYY (SEQ ID NO: 202) GLP-1⁹Gln-GLP-1(7-37) (SEQ ID NO: 168), D-⁹Gln-GLP-1(7-37) (SEQ IDNO: 169), ¹⁶Thr-¹⁸ Lys ⁻GLP-1(7-37) (SEQ ID NO: 170), ¹⁸Lys-GLP-1(7-37) (SEQ ID NO: 171), ⁸Gly-GLP-1 (7-36) (SEQ ID NO: 172), ⁹Gln-GLP-1(7-37) (SEQ ID NO: 173), D-⁹Gln-GLP-1 (7-37) (SEQ ID NO: 174),acetyl-⁹Lys-GLP-1(7-37) (SEQ ID NO: 175), ⁹Thr-GLP-1(7-37) (SEQ IDNO: 176), D-⁹Thr-GLP-1 (7-37) (SEQ ID NO: 177), ⁹Asn-GLP-1 (7-37)(SEQ ID NO: 178), D-⁹Asn-GLP-1 (7-37) (SEQ ID NO: 179),²²Ser²³Arg²⁴Arg²⁶Gln-GLP-1(7-37) (SEQ ID NO: 180), ¹⁶Thr¹⁸Lys-GLP-1(7-37) (SEQ ID NO: 181), ¹⁸Lys-GLP-1(7-37) (SEQ ID NO: 182), ²³Arg-GLP-1(7-37) (SEQ ID NO: 183), ²⁴Arg-GLP-1(7-37) (SEQ ID NO: 184) Exendin¹⁴Leu,²⁵Phe-exendin-4 (SEQ ID NO: 185), ⁵Ala,¹⁴Leu,²⁵Phe-exendin-4(SEQ ID NO: 186), and ¹⁴Leu,²²Ala,²⁵Phe-exendin-4 (SEQ ID NO: 187).

As known in the art, such peptide compounds may preferably be amidated,but within the context of the present invention, may optionally be inthe acid form unless otherwise specified.

Still other preferred bioactive peptide hormone modules includefragments of a component peptide hormone selected from: amylin, ADM, CT,CGRP, intermedin, CCK, leptin, PYY(1-36) (SEQ ID NO:57), PYY(3-36) (SEQID NO:58), GLP-1(1-37) (SEQ ID NO:59), GLP-1(7-37) (SEQ ID NO:204),GLP-1(7-36) (SEQ ID NO:61), GLP-2, OXM, a natriuretic peptide,exendin-3, and exendin-4, urocortin family peptides, e.g., Ucn-2 andUcn-3, neuromedin family peptides, e.g. neuromedin U25 or splicevariants, and ANP, BNP, CNP or urodilatin wherein the fragment exhibitsat least one hormonal activity of the component peptide hormone.

Yet other preferred bioactive peptide hormone modules include fragmentsof analogs or derivatives of a component peptide hormone selected from:amylin, ADM, CT, CGRP, intermedin, CCK, leptin, PYY(1-36) (SEQ IDNO:57), PYY(3-36) (SEQ ID NO:58), GLP-1(1-37) (SEQ ID NO:59),GLP-1(7-37) (SEQ ID NO:204), GLP-1(7-36) (SEQ ID NO:61), GLP-2, OXM,ANP, BNP, CNP, urodilatin, exendin-3, exendin-4, a natriuretic peptidehormones, urocortin family peptides, e.g., Ucn-2 and Ucn-3, neuromedinfamily peptides, e.g. neuromedin U25 or splice variants, and ANP, BNP,CNP or urodilatin, wherein the fragment exhibits at least one hormonalactivity of the component peptide hormone. Again, the analog maycomprise one or more insertions, deletions, or substitutions of theamino acid sequence of the component peptide hormone, and the derivativemay comprise one or more chemical modifications of an amino acid residueof an analog or component peptide hormone, as described more fullyherein and known in the art.

Certain preferred fragments that exhibit at least one hormonal activityinclude the following. However, it should be understood thatcombinations of the above-described analogs and derivatives taken withfragments known in the art, including the preferred fragments describedbelow, are contemplated.

Amylin: amylin(1-36) (SEQ ID NO: 210), amylin(1-35) (SEQ ID NO: 211),amylin(1-20) (SEQ ID NO: 212), amylin(1-18) (SEQ ID NO: 213),amylin(1-17) (SEQ ID NO: 214), amylin (1-16) (SEQ ID NO: 215),amylin(1-15) (SEQ ID NO: 216), amylin(1-7) (SEQ ID NO: 217) CT: CT(8-32)(SEQ ID NO: 218), CT(8-27) (SEQ ID NO: 219), CT(8-26) (SEQ ID NO: 220),CT(8-10) (SEQ ID NO: 221), CT(18-26) (SEQ ID NO: 222), CT(18-27) (SEQ IDNO: 223) AFP-6: AFP-6(18-27) (SEQ ID NO: 224) CCK: CCK-8, CCK-5, CCK-4Leptin: leptin (22-167) (SEQ ID NO: 225), leptin(56-73) (SEQ ID NO: 226)PYY: PYY(1-35) (SEQ ID NO: 227), PYY(1-30) (SEQ ID NO: 228), PYY(1-25)(SEQ ID NO: 229), PYY(1-15) (SEQ ID NO: 230), PYY(1-10) (SEQ ID NO:231), PYY(2-36) (SEQ ID NO: 232), PYY(3-36) (SEQ ID NO: 58), PYY(4-36)(SEQ ID NO: 233), PYY(5-36) (SEQ ID NO: 234) GLP-1 GLP-1(7-37) (SEQ IDNO: 204), GLP-1(7-36) (SEQ ID NO: 61), GLP-1(7-35) (SEQ ID NO: 235)Exendin exendin-4(1-27) (SEQ ID NO: 236), exendin-4(1-28) (SEQ ID NO:237), exendin-4(1-29) (SEQ ID NO: 238), exendin-4(1-30) (SEQ ID NO: 239)or longer

Again, as known in the art, such peptide compounds may preferably beamidated, but within the context of the present invention, mayoptionally be in the acid form unless otherwise specified. Further, theabove preferred fragments may be combined with any of the analogs orderivatives discussed herein or known in the art. For example, preferredanalog fragments may include ⁵Ala,¹⁴Leu,²⁵Phe-exendin-4(1-28) (SEQ IDNO:240), ¹⁴Leu,²⁵Phe-exendin-4(1-27) (SEQ ID NO:241),⁵Ala,¹⁴Leu,²⁵Phe-exendin-4(1-28) (SEQ ID NO:240),¹⁴Leu,²⁵Phe-exendin-4(1-27) (SEQ ID NO:241), or any other combinationsof the disclosed fragments, analogs, and derivatives.

Yet other preferred bio-active peptide modules include “peptidicenhancer”, i.e., structural motifs of component peptide hormones(including analogs and derivatives thereof) that impart a desiredchemical stability, conformational stability, metabolic stability,bioavailability, organ/tissue targeting, receptor interaction, proteaseinhibition, plasma protein binding, and/or other pharmacokineticcharacteristic to the hybrid polypeptide. Exemplary peptidic enhancersinclude the following. Again, it should be understood that combinationsof the above-described analogs and derivatives taken together with thefollowing bio-active peptide modules are contemplated. For example, thelast six amino acid residues of amylin family peptide hormone analogsand derivatives known in the art and/or described above are alsocontemplated as preferred bio-active peptide modules.

Amylin Family amylin(32-37) (SEQ ID NO: 242), amylin(33-37) (SEQ ID NO:243), amylin(34-37) (SEQ ID NO: 244), amylin(35-37), amylin(36-37),amylin(37), ADM(47-52) (SEQ ID NO: 245), ADM(48-52) (SEQ ID NO: 246),ADM(49-52) (SEQ ID NO: 247), ADM(50-52), ADM(51-52), ADM(52), CT(27-32)(SEQ ID NO: 248), CT(27-32) (SEQ ID NO: 249), CT(28-32) (SEQ ID NO:250), CT(29-32), CT(30-32), CT(31-32), CT(32), CGRP(32-37) (SEQ ID NO:251), CGRP(33-37) (SEQ ID NO: 252), CGRP(34-37) (SEQ ID NO: 253),CGRP(35-37), CGRP(36-37), CGRP(37), intermedin (42-47) (SEQ ID NO: 254),intermedin (43-47) (SEQ ID NO: 255), intermedin (44-47) (SEQ ID NO:256), intermedin (45-47), intermedin (46-47), intermedin (47) PYYPYY(25-36) (SEQ ID NO: 257), PYY(26-36) (SEQ ID NO: 258), PYY(27-36)(SEQ ID NO: 259), PYY(28-36) (SEQ ID NO: 260), PYY(29-36) (SEQ ID NO:261), PYY(30-36) (SEQ ID NO: 262), PYY(31-36) (SEQ ID NO: 263),PYY(32-36) (SEQ ID NO: 264), PYY(25-35) (SEQ ID NO: 265), PYY(26-35)(SEQ ID NO: 266), PYY(27-35) (SEQ ID NO: 267), PYY(28-35) (SEQ ID NO:268), PYY(29-35) (SEQ ID NO: 269), PYY(30-35) (SEQ ID NO: 270),PYY(31-35) (SEQ ID NO: 271), PYY(32-35) (SEQ ID NO: 272) GLP-1 and 2frog GLP-1(29-37) (SEQ ID NO: 273); frog GLP-1(30-37) (SEQ ID NO: 273);frog GLP-2(24-31) (SEQ ID NO: 275), frog GLP-2(25-31) (SEQ ID NO: 276)Exendin-4 exendin-4(31-39) (SEQ ID NO: 277), exendin-4(32-39) (SEQ IDNO: 278), exendin-4(33-39) (SEQ ID NO: 279), exendin-4(34-39) (SEQ IDNO: 280), exendin-4(35-39) (SEQ ID NO: 281), exendin- 4(36-39) (SEQ IDNO: 282), exendin-4(37-39), exendin-4(38-39), exendin-4(39)

8. Peptide Module Selection Considerations, Spacers, and Linking Groups

The hybrid polypeptides of the present invention generally comprise atleast two bio-active peptide hormone modules of the invention, whereinat least one of the bio-active peptide hormone modules exhibits at leastone hormonal activity. The bio-active peptide hormone module thatexhibits the at least one hormonal activity may be located at theN-terminal end of the hybrid polypeptide, the C-terminal end of thehybrid polypeptide, or in the event that the hybrid polypeptidecomprises more than two bio-active peptide hormone modules, may belocated in the internal portion of the hybrid polypeptide.

In certain embodiments, it may be preferable to locate the bio-activepeptide hormone module exhibiting the at least one hormonal activitysuch that the C-terminal end of the bio-active peptide hormone module isamidated. Amidation of the C-terminal end of the bio-active peptidehormone module may be accomplished by locating the module at theC-terminal end of the hybrid peptide, or by configuring the module inthe C-terminal-to-N-terminal direction at the N-terminal end of thehybrid polypeptide. In both configurations, the C-terminal end of thebio-active peptide hormone module is available for amidation. Specificcomponent peptide hormones where C-terminal amidation may be preferableinclude amylin family peptide hormones, CCK, PYY, hGLP-1(7-36) (SEQ IDNO:61), and hGLP-2. Specific component peptide hormones where C-terminalamidation is not necessarily preferred (stated otherwise, whereelongation at the C-terminal end of the module is easily tolerated)include exendin-4, exendin-4(1-28) (SEQ ID NO:237), GLP-1(7-37) (SEQ IDNO:204), frog GLP-1(7-36) (SEQ ID NO:283), and frog GLP-2. However, ifthese component peptide hormones are located at the C-terminal end ofthe hybrid polypeptide, they may still be optionally amidated, and infact may preferably be optionally amidated.

The bio-active peptide hormone modules may be covalently linked in anymanner known in the art. Stable linkages may be used, or cleavablelinkage may be used. In one embodiment, the carboxy of a first modulemay be directly linked to the amino of a second module. In anotherembodiment, linking groups may be used to attached modules. Further, ifdesired, spacers or turn inducers known in the art may be employed tostabilize the linkage. By way of example, where amidation of theC-terminal end of the N-terminally located bio-active peptide hormonemodule is not desired, the module may be attached to a second moduledirectly, or using any appropriate linking group known in the art, suchas, an alkyl; PEG; amino acid, e.g., Lys, Glu, 13-Ala; polyaminoacids,e.g., poly-his, poly-arg, poly-lys, poly-ala, Gly-Lys-Arg (GKR) etc.;bifunctional linker (see, e.g., Pierce catalog, Rockford, Ill.);aminocaproyl (“Aca”), β-alanyl, 8-amino-3,6-dioxaoctanoyl, or othercleavable and non-cleavable linker known in the art. Specificallydescribed herein, as if each were explicitly drawn, are embodiments ofspecific hybrids in which the linker in each exemplifiedlinker-containing hybrid is replaced by a Gly linker, particularlyembodiments where the Gly linker is Gly-Gly-Gly. As an example, forexemplified species ²⁹5Apa-Exendin(1-28)-¹des-Lys-hAmylin(1-7)-^(11,18)Arg-sCt(8-27)-hAmylin(33-37)(SEQ ID NO:32) (see tables herein) its Gly linker species analog is alsospecifically intended and disclosed. This species is²⁹GlyGlyGly-Exendin(1-28)-¹des-Lys-hAmylin(1-7)-^(11,18)Arg-sCt(8-27)-hAmylin(33-37)(SEQ ID NO:313), where the three glycines are located after theexendin(1-28) sequence. In one embodiment a linker or spacer is 1 to 30residues long, in another embodiment 2 to 30 residues, and in yetanother 3-30 residues long, and any integer length from 2 to 30inclusive; each integer unit is contemplated, e.g. 2, 3, 4, 5, 6, 7,etc. In one embodiment a Gly linker is used, and in a particularembodiment a three residue linker Gly-Gly-Gly.

Where amidation of the C-terminal end of N-terminally located bio-activepeptide hormone module is desired, the module may again be attached to asecond module using any appropriate linking group known in the art. Morespecifically, in the event that a bio-active peptide hormone moduleexhibiting at least one hormonal activity has been configured in theC-terminal-to-N-terminal orientation, resulting in an amino to aminolinkage, preferred linking groups include dicarboxylic acids, alkyls,PEGs, and amino acids such as Lys, Cys, and Glu.

As mentioned above, the hybrid polypeptides may also preferably includespacer to further stablize the linkage of the bio-active peptide hormonemodules. Any spacer or turn inducer known in the art may be used. By wayof example, referred β-turn mimetics include mimic A and mimic Billustrated below, also Ala-Aib and Ala-Pro dipeptides. Their IUPACnames are Mimic A:N-(3S,6S,9S)-2-oxo-3-amino-1-azabicyclo[4.3.0]-nonane-9-carboxylic acid.Mimic B:N-(3S,6S,9R)-2-oxo-3-amino-7-thia-1-azabicyclo[4.3.0]-nonane-9-carboxylicacid.

9. Exemplary Combinations and Specific Embodiments

Exemplary combinations of bio-active peptide hormone modules to form thehybrid polypeptides of the invention include combinations of two or morebio-active peptide hormone modules selected from: native peptidehormones, analogs and derivatives of peptide hormones that exhibit atleast one hormonal activity, fragments of native peptide hormones thatexhibit at least one hormonal activity, fragments of analogs andderivatives of peptides hormones that exhibit at least one hormonalactivity, and peptidic enhancers, with the proviso that at least onemodule exhibit at least one hormonal activity.

The hybrid polypeptides of the invention will include at least twobio-active peptide hormone modules, wherein each module is comprisedfrom component peptide hormones. In the context of the presentinvention, the component peptide hormones of the hybrid polypeptide maybe the same or different, with the proviso that at least two of thecomponent peptide hormones are different. In a preferred embodiment, atleast two of the component peptide hormones are from different peptidehormone families, e.g., the amylin family, CCK, the leptin family, PPF,the proglucagon family, the natriuretic peptide family, urocortin familypeptides, e.g., Ucn-2 and Ucn-3, neuromedin family peptides, e.g.neuromedin U25 or splice variants, and ANP, BNP, CNP or urodilatin andthe GLP-1 and exendin family.

In certain embodiments, the hybrid polypeptides of the invention maycomprise two or more modules that exhibit at least one hormonalactivity. For instance, the hybrid polypeptide may comprise a fragmentof a first peptide hormone or analog that exhibits at least one hormonalactivity covalently, linked to a fragment of at least one additionalpeptide hormone analog. The additional fragment(s) may optionallyexhibit at least one hormonal activity. The first peptide hormone may bethe same or different from the additional peptide hormone(s), with theproviso that at least one of the additional peptide hormones aredifferent from the first peptide hormone, and the first hormonalactivity may be the same or different from the optional additionalhormonal activity.

In other embodiments, the hybrid polypeptides of the invention maycomprise one or more modules that exhibit at least one hormonal activityin combination with one or more peptidic enhancer modules. For instance,a fragment of a first peptide hormone that exhibits a at least onehormonal activity may be covalently linked to a peptidic enhancer, or afragment of a first peptide hormone that exhibits at least one hormonalactivity may be covalently linked to a second peptide hormone thatexhibits at least one hormonal activity, which is in turn linked to apeptidic enhancer. Alternatively, a peptidic enhancer may be locatedbetween two peptide hormone modules as a stabilizing spacer. Again, thefirst peptide hormone may be the same or different from the secondpeptide hormone, and the first hormonal activity may be the same ordifferent from the second hormonal activity.

In another embodiment, the hybrid polypeptides of the invention maycomprise two, three, four, or more bio-active peptide hormone modules.Exemplary combinations include a module with a hormonal activity incombination with one, two, or three peptidic enhancers; two modules witha hormonal activity in combination with one or two peptidic enhancers;three modules with a hormonal activity in combination with one peptidicenhancer, etc.

The component peptide hormones are preferably selected from amylin,adrenomedullin, calcitonin, calcitonin gene related peptide, intermedin,cholecystokinin, leptin peptide YY, glucagon-like peptide-1,glucagon-like peptide 2, oxyntomodulin, ANP, BNP, CNP, urodilatin,natriuretic peptide hormones, urocortin family peptides, e.g., Ucn-2 andUcn-3, neuromedin family peptides, e.g. neuromedin U25 or splicevariants, and ANP, BNP, CNP or urodilatin or exendin-4.

More particularly, preferred module combinations include those involvingcombinations of exendin, amylin (and/or sCT), BNP, and PYY as thecomponent peptide hormones. Particular combinations includeexendin-4/PYY and PYY/exendin-4 combinations, with and without spacersor linking groups. Other combinations include exendin/amylin andamylin/exendin combinations, with and without spacers or linking groups.Yet other combinations include amylin/PYY and PYY/amylin combinations,with and without spacers or linking groups.

In one aspect, preferred module combinations include those involving afirst module comprising exendin-4, a fragment of exendin-4 that exhibitsat least one hormonal activity, an exendin-4 analog or derivative thatexhibits at least one hormonal activity, or a fragment of an exendin-4analog that exhibits at least one hormonal activity in combination withat least one additional bio-active peptide hormone module. In oneembodiment, the first module is linked to one, two, or three additionalbio-active peptide hormone modules.

In preferred embodiments, a first module comprising an exendin-4 peptideis linked to a second bio-active peptide hormone module comprising anamylin (and/or sCT) peptide that exhibits at least one hormonalactivity. In another embodiment, the second module is further linked toa third bio-active peptide hormone module comprising a calcitoninpeptide that exhibits at least one hormonal activity. In yet anotherembodiment, the third module may be further linked to a fourthbio-active peptide hormone module comprising a peptidic enhancerselected from amylin peptides. In one embodiment, the first module maybe located at the C-terminal end of the hybrid polypeptide.Alternatively, the first module may be located at the N-terminal end ofthe hybrid polypeptide. In certain embodiments, spacers or linkers suchas βAla may be inserted if desired to link the modules.

Preferred exendin-4 peptides include: exendin-4, exendin-4(1-27) (SEQ IDNO:236), exendin-4(1-28) (SEQ ID NO:237), ¹⁴Leu,²⁵Phe-exendin-4(1-28)(SEQ ID NO:284), and ⁵Ala,¹⁴Leu,²⁵Phe-exendin-4(1-28) (SEQ ID NO:240).Also useful are exendin(7-15) and its Ser2 analog, HSEGTFTSD (SEQ IDNO.378). Preferred amylin peptides that exhibit at least one hormonalactivity include amylin, amylin fragments such as amylin(1-17) (SEQ IDNO:214), amylin (1-16) (SEQ ID NO:215), amylin(1-15) (SEQ ID NO:216),and amylin(1-7) (SEQ ID NO:217), and amylin analogs such as pramlintide,²Ala-h-amylin, ^(2,7)Ala-h-amylin (SEQ ID NO:79), and fragments thereof.Preferred calcitonin peptides that exhibit at least one hormonalactivity sCT, sCT fragments such as sCT(8-10), sCT(8-27) (SEQ IDNO:288), and calcitonin analogs such as ^(11,18)Arg-sCT (SEQ ID NO:108),¹⁸Arg-sCT (SEQ ID NO:107), ¹⁴Glu,¹⁸Arg-sCT (SEQ ID NO:109),¹⁴Glu,^(11,18)Arg-sCT (SEQ ID NO:110), and fragments thereof. Preferredamylin peptidic enhancers include amylin(32-37) (SEQ ID NO:242),amylin(33-37) (SEQ ID NO:243), and amylin(34-37) (SEQ ID NO:244), andanalogs thereof. Amylin/sCT combinations useful in connection with thepresent invention include those disclosed in PCT/US2005/004631 AmylinFamily Agonist, Attorney Docket 18528.835, which is herein incorporatedby reference. An amylin/sCT chimera particularly useful for creatinghybrids of the invention is Compound 10 (described herein and inPCT/US2005/004631) and analogs and derivatives thereof.

In one aspect, preferred module combinations include those involving afirst module comprising exendin-4, a fragment of exendin-4 that exhibitsat least one hormonal activity, an exendin-4 analog or derivative thatexhibits at least one hormonal activity, or a fragment of an exendin-4analog that exhibits at least one hormonal activity in combination witha peptidic enhancer. Preferred exendin-4 compounds include: exendin-4,exendin-4(1-27) (SEQ ID NO:236), exendin-4(1-28) (SEQ ID NO:237),¹⁴Leu,²⁵Phe-exendin-4(1-28) (SEQ ID NO:284), and⁵Ala,¹⁴Leu,²⁵Phe-exendin-4(1-28) (SEQ ID NO:240). Preferred peptidicenhancers include: PYY(25-36) (SEQ ID NO:257), PYY(30-36) (SEQ IDNO:262) and PYY(31-36) (SEQ ID NO:263). In one embodiment, the firstmodule is located at the C-terminal end of the hybrid polypeptide andthe peptidic enhancer is located at the N-terminal end of the hybridpolypeptide. Alternatively, the first module may be located at theN-terminal end of the hybrid polypeptide and the peptidic enhance may belocated at the C-terminal end of the hybrid polypeptide. In certainembodiments, spacers or linkers such as βAla may be inserted if desiredto attach the modules.

In another aspect, preferred module combinations include those involvinga first module comprising exendin-4, a fragment of exendin-4 thatexhibits at least one hormonal activity, an exendin-4 analog orderivative that exhibits at least one hormonal activity, or a fragmentof an exendin-4 analog that exhibits at least one hormonal activity incombination with a second module comprising CCK, a fragment of CCK thatexhibits at least one hormonal activity, a CCK analog or derivative thatexhibits at least one hormonal activity, or a fragment of a CCK analogthat exhibits at least one hormonal activity. Again, preferred exendin-4compounds include: exendin-4, exendin-4(1-27) (SEQ ID NO:236),exendin-4(1-28) (SEQ ID NO:237), ¹⁴Leu,²⁵Phe-exendin-4(1-28) (SEQ IDNO:284), ⁵Ala,¹⁴Leu,²⁵Phe-exendin-4(1-28) (SEQ ID NO:240), and¹⁴Leu-exendin-4(1-28) (SEQ ID NO:190). Preferred CCK compounds include:CCK-8, and CCK-8(Phe(CH₂SO₃)). In one embodiment, the first module islocated at the C-terminal end of the hybrid polypeptide and the secondmodule is located at the N-terminal end of the hybrid polypeptide.Alternatively, the first module may be located at the N-terminal end ofthe hybrid polypeptide and the peptidic enhance may be located at theC-terminal end of the hybrid polypeptide. In certain embodiments,spacers or linkers such as βAla may be inserted if desired to attach themodules.

In another aspect, preferred module combinations include those involvinga first module comprising amylin, a fragment of amylin that exhibits atleast one hormonal activity, an amylin analog or derivative thatexhibits at least one hormonal activity, or a fragment of an amylinanalog that exhibits at least one hormonal activity in combination witha second module comprising with a peptidic enhancer, such as PYY(25-36)(SEQ ID NO:257) or PYY(30-36) (SEQ ID NO:262). In one embodiment, thefirst module is located at the C-terminal end of the hybrid polypeptideand the peptidic enhancer is located at the N-terminal end of the hybridpolypeptide. Alternatively, the first module may be located at theN-terminal end of the hybrid polypeptide and the peptidic enhance may belocated at the C-terminal end of the hybrid polypeptide. In certainembodiments, spacers or linkers such as βAla may be inserted if desiredto attach the modules.

Other preferred module combinations include those involving combinationsof exendin and CCK or amylin, calcitonin, and CCK as a tertiarycombination. Particular combinations include exendin/CCK andCCK/exendin, with and without spacers or linkers and linking groups.Other combinations include CCK/amylin/calcitonin andCCK/amylin/calcitonin/amylin, with and without spacers or linkinggroups. Each module may independently be a peptidic enhancer or mayexhibit a hormonal activity, depending on the desired properties of thehybrid polypeptide.

Yet other preferred module combinations include those involvingcombinations of exendin, amylin and calcitonin as tertiary andtetra-hybrid molecules. Exemplary combinations includeexendin/amylin/calcitonin; exendin/amylin/calcitonin/amylin;amylin/calcitonin/exendin; and amylin/calcitonin/amylin/exendincombinations, with and without spacers or linking groups. Each modulemay independently be a peptidic enhancer or may exhibit a hormonalactivity, depending on the desired properties of the hybrid polypeptide.

In one embodiment, when one of the bio-active peptide hormone module(s)that exhibits at least one hormonal activity is amylin or an analog orfragment thereof, and a second bio-active peptide hormone modulecomprises CCK, then the hybrid polypeptide should preferably comprise athird bio-active peptide hormone module selected from a differentcomponent peptide hormone. Exemplary third bio-active peptide hormonemodules include calcitonins, more preferably salmon calcitonin, analogsor fragments thereof.

In another embodiment, when one of the bio-active peptide hormonemodule(s) that exhibits at least one hormonal activity is amylin or ananalog or fragment thereof, and a second bio-active peptide hormonemodule comprises CT, then the hybrid polypeptide should preferablycomprise a third bio-active peptide hormone module selected from adifferent component peptide hormone. Exemplary third bio-active peptidehormone modules include exendin-4, analogs or fragments thereof.

In yet another embodiment, when one of the bio-active peptide hormonemodule(s) that exhibits at least one hormonal activity is GLP-1 or ananalog or fragment thereof, and a second bio-active peptide hormonemodule is a peptidic enhancer comprising an exendin fragment, then thehybrid polypeptide should preferably comprise a third bio-active peptidehormone module. Exemplary third bio-active peptide hormone modulesinclude PYY (including analogs, derivatives and fragments thereof) andCCK (including analogs, derivatives and fragments thereof).

Within each of the combinations described herein, it is understood thatreference to a component peptide hormone includes reference to analogs,derivatives, fragments, as well as peptidic enhancers related thereto.

In a preferred aspect, the hybrid polypeptides include:

SEQ ID NO: 1 Exendin-4-PYY(22-36) 2 Exendin-4-PYY(25-36) 3Exendin-4-PYY(18-36) 4 Exendin-4-βAla-βAla-PYY(22-36) 5Exendin-4-βAla-βAla-PYY(25-36) 6 Exendin-4-βAla-βAla-PYY(31-36) 7Exendin-4(1-28)-PYY(22-36) 8 Exendin-4(1-28)-PYY(25-36) 9Exendin-4(1-28)-PYY(18-36) 10 Exendin-4(1-28)-βAla-βAla-PYY(22-36) 11Exendin-4(1-28)-βAla-βAla-PYY(25-36) 12Exendin-4(1-28)-βAla-βAla-PYY(31-36) 13⁵Ala,¹⁴Leu,²⁵Phe-Exendin-4(1-28)-PYY(18-36) 14⁵Ala,¹⁴Leu,²⁵Phe-Exendin-4(1-28)-PYY(22-36) 15⁵Ala,¹⁴Leu,²⁵Phe-Exendin-4(1-28)-PYY(25-36) 16⁵Ala,¹⁴Leu,²⁵Phe-Exendin-4(1-17)-PYY(18-36) 17⁵Ala,¹⁴Leu,²⁵Phe-Exendin-4(1-28)-βAla-βAla-PYY(22-36) 18⁵Ala,¹⁴Leu,²⁵Phe-Exendin-4(1-28)-βAla-βAla-PYY(25-36) 19⁵Ala,¹⁴Leu,²⁵Phe-Exendin-4(1-28)-βAla-βAla-PYY(31-36) 20 Exendin-4-CCK-821 Exendin-4(1-28)-CCK-8 22 Exendin-4(1-28)-CCK-8(Phe(CH₂SO₃)) 23Exendin-4(1-28)-(8-amino-3,6-dioxactoanoyl)-CCK-8 24Exendin-4(1-28)-(8-amino-3,6-dioxactoanoyl)-CCK-8(Phe(CH₂SO₃)) 25Exendin-4(1-27)-hAmylin(1-7)-^(11,18)Arg-sCT(8-27)-Amylin(33-37) 26Exendin-4(1-27)-^(2,7)Ala-hAmylin(1-7)-sCT(8-10) 27 ²⁹12Ado-Exendin(1-28)-hAmylin(1-7)-^(11,18)Arg-sCt(8-27)-hAmylin(33-37) 28²⁹12 Ado-Exendin(1-28)-¹des-Lys-hAmylin(1-7)-^(11,18)Arg-sCt(8-27)-hAmylin(33-37) 29²⁹3,6-dioxaoctanoyl-Exendin(1-28)-hAmylin(1-7)-^(11,18)Arg-sCt(8-27)-hAmylin(33-37) 30²⁹3,6-dioxaoctanoyl-Exendin(1-28)-¹des-Lys-hAmylin(1-7),^(11,18)Arg-sCt(8-27)-hAmylin(33-37) 31 ²⁹5Apa-Exendin(1-28)-hAmylin(1-7)-^(11,18)Arg-sCt(8-27)-hAmylin(33-37) 32²⁹5 Apa⁻Exendin(1-28)-¹des-Lys-hAmylin(1-7)-^(11,18)Arg-sCt(8-27)-hAmylin(33-37) 33²⁹βAla-βAla-Exendin(1-28),hAmylin(1-7)-^(11,18)Arg-sCt(8-27)-hAmylin(33-37) 34²⁹βAla-βAla-Exendin(1-28)-¹des-Lys-hAmylin(1-7)-^(11,18)Arg-sCt(8-27)-hAmylin(33-37) 35 ²⁹4,7,10-trioxa-13-tridecanaminesuccinimidyl-Exendin(1-28)-hAmylin(1-7)^(11,18)Arg-sCt(8-27)-hAmylin(33-37) 36²⁹4,7,10-trioxa-13-tridecanamine succinimidyl-Exendin(1-28)-¹des-Lys-hAmylin(1-7)-^(11,18)Arg-sCt(8-27)-hAmylin(33-37) 37CCK-8-GKR-¹⁵Glu-hAmylin(1-17)-¹⁸Arg-sCT(18-26)-Amylin(32-37) 38Amylin(1-18)-PYY(19-36) 39isocaproyl-STAVL-(Aib)-K(formyl)-LSQEL-(Aib)-K(formyl)-LQT- PYY(18-36)40 isocaproyl-STAVL-(Aib)-K(formyl)-LSQEL-(Aib)-K(formyl)-L- PYY(16-36)41 CCK-8-[Succinoyl-Cys]-PYY(3-36) 42CCK-8-[Bis-Cys(N-Acetyl)]-PYY(3-36) 43CCK-8-[Gly-Aminoxymethylcarbonyl]-PYY(3-36) 379Exendin-4(1-27)-hAmylin(1-7)-¹⁴Glu,^(11,18)Arg-sCT(8-27)-Amylin(33-37)380 ²⁹12 Ado-Exendin(1-28)-hAmylin(1-7)-14Glu,^(11,18)Arg-sCt(8-27)-hAmylin(33-37) 381 ²⁹12Ado-Exendin(1-28)-¹des-Lys-hAmylin(1-7)-¹⁴Glu,^(11,18)Arg-sCt(8-27)-hAmylin(33-37) 382²⁹3,6-dioxaoctanoyl-Exendin(1-28)-hAmylin(1-7)-¹⁴Glu,^(11,18)Arg-sCt(8-27)-hAmylin(33-37) 383²⁹3,6-dioxaoctanoyl-Exendin(1-28)-¹des-Lys-hAmylin(1-7),¹⁴Glu,^(11,18)Arg-sCt(8-27)-hAmylin(33-37) 384 ²⁹5Apa-Exendin(1-28)-hAmylin(1-7)-¹⁴Glu,^(11,18)Arg-sCt(8-27)-hAmylin(33-37) 385 ²⁹5Apa⁻Exendin(1-28)-¹des-Lys-hAmylin(1-7)-¹⁴Glu,^(11,18)Arg-sCt(8-27)-hAmylin(33-37) 386²⁹βAla-βAla-Exendin(1-28),hAmylin(1-7)-¹⁴Glu,^(11,18)Arg-sCt(8-27)-hAmylin(33-37) 387²⁹βAla-βAla-Exendin(1-28)-¹des-Lys-hAmylin(1-7)-¹⁴Glu,^(11,18)Arg-sCt(8-27)-hAmylin(33-37) 388 ²⁹4,7,10-trioxa-13-tridecanaminesuccinimidyl-Exendin(1-28)-hAmylin(1-7)-¹⁴Glu,^(11,18)Arg-sCt(8-27)-hAmylin(33-37) 389²⁹4,7,10-trioxa-13-tridecanamine succinimidyl-Exendin(1-28)-¹des-Lys-hAmylin(1-7)-¹⁴Glu,^(11,18)Arg-sCt(8-27)-hAmylin(33-37) 390CCK-8-GKR-¹⁵Glu-hAmylin(1-17)-¹⁴Glu,¹⁸Arg-sCT(18-26)- Amylin(32-37)

Exemplary exendin and neuromedin hybrids include

Exendin-(1-28)-beta-Ala-beta-Ala-FN-38: (SEQ ID NO: 391)HGEGTFTSDLSKQMEEEAVRLFIEWLKN-beta-Ala-beta-Ala-FLFHYSKTQKLGKSNVVEELQSPFASQSRGYFLFRPRN-NH2);Exendin-(1-28)-beta-Ala-beta-Ala-Neuromedin(U25): (SEQ ID NO: 392)HGEGTFTSDLSKQMEEEAVRLFIEWLKN-beta-Ala-beta-Ala-FRVDEEFQSPFASQSRGYFLFRPRN-NH2; andExendin-(1-28)-beta-Ala-beta-Ala-Neuromedin(U-9): (SEQ ID NO: 393)HGEGTFTSDLSKQMEEEAVRLFIEWLKN-beta-Ala-beta-Ala- GYFLFRPRN-NH2

The beta-Ala-beta-Ala spacer is optional, and can be replaced withGly-Gly-Gly, a mini-PEG group, or other linker known in the art,particularly those described herein.

Exemplary exendin and natriuretic peptide hybrids include exendin-hBNPpeptide hybrids, including

Exendin-(1-28)-beta-Ala-beta-Ala-hBNP: (SEQ ID NO: 394)HGEGTFTSDLSKQMEEEAVRLFIEWLKN-beta-Ala-beta-Ala-SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH; and Exendin-beta-Ala-beta-Ala-hBNP:(SEQ ID NO: 395) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-β-Ala-β-Ala-SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH.

As in all of the hybrids of the invention, a beta-Ala-beta-Ala spacer isoptional, and can be replaced with Gly-Gly-Gly, a mini-PEG group, orother linker known in the art, particularly those described herein.

The hybrid polypeptides of the present invention may also comprisefurther modifications including, but are not limited to, substitution,deletion, and insertion to the amino acid sequence of such hybridpolypeptides and any combination thereof. In a preferred aspect, thehybrid polypeptides of the invention include one or more modificationsof a “non-essential” amino acid residue. In the context of theinvention, a “non-essential” amino acid residue is a residue that can bealtered, i.e., deleted or substituted, in the native human amino acidsequence of the fragment, e.g., the component peptide hormone fragment,without abolishing or substantially reducing the component peptidehormone receptor agonist activity of the hybrid polypeptide.

Preferred substitutions include conserved amino acid substitutions. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain, or physicochemical characteristics (e.g., electrostatic, hydrogenbonding, isosteric, hydrophobic features). Families of amino acidresidues having similar side chains are known in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, methionine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,tryptophan), β-branched side chains (e.g., threonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine).

The present invention also relates to derivatives of the hybridpolypeptides. Such derivatives include hybrid polypeptides conjugated toone or more water soluble polymer molecules, such as polyethylene glycol(“PEG”) or fatty acid chains of various lengths (e.g., stearyl,palmitoyl, octanoyl, etc.), or by the addition of polyamino acids, suchas poly-his, poly-arg, poly-lys, and poly-ala. Modifications to thehybrid polypeptides can also include small molecule substituents, suchas short alkyls and constrained alkyls (e.g., branched, cyclic, fused,adamantyl), and aromatic groups. The water soluble polymer moleculeswill preferably have a molecular weight ranging from about 500 to about20,000 Daltons.

Such polymer-conjugations and small molecule substituent modificationsmay occur singularly at the N- or C-terminus or at the side chains ofamino acid residues within the sequence of the hybrid polypeptides.Alternatively, there may be multiple sites of derivatization along thehybrid polypeptide. Substitution of one or more amino acids with lysine,aspartic acid, glutamic acid, or cysteine may provide additional sitesfor derivatization. See, e.g., U.S. Pat. Nos. 5,824,784 and 5,824,778.Preferably, the hybrid polypeptides may be conjugated to one, two, orthree polymer molecules.

The water soluble polymer molecules are preferably linked to an amino,carboxyl, or thiol group, and may be linked by N or C terminus, or atthe side chains of lysine, aspartic acid, glutamic acid, or cysteine.Alternatively, the water soluble polymer molecules may be linked withdiamine and dicarboxylic groups. In a preferred embodiment, the hybridpolypeptides of the invention are conjugated to one, two, or three PEGmolecules through an epsilon amino group on a lysine amino acid.

Hybrid polypeptide derivatives of the invention also include hybridpolypeptides with chemical alterations to one or more amino acidresidues. Such chemical alterations include amidation, glycosylation,acylation, sulfation, phosphorylation, acetylation, and cyclization. Thechemical alterations may occur singularly at the N- or C-terminus or atthe side chains of amino acid residues within the sequence of the PPFhybrid polypeptides. In one embodiment, the C-terminus of these peptidesmay have a free —OH or —NH₂ group. In another embodiment, the N-terminalend may be capped with an isobutyloxycarbonyl group, anisopropyloxycarbonyl group, an n-butyloxycarbonyl group, anethoxycarbonyl group, an isocaproyl group (isocap), an octanyl group, anoctyl glycine group (G(Oct)), or an 8-aminooctanic acid group. In apreferred embodiment, cyclization can be through the formation ofdisulfide bridges. Alternatively, there may be multiple sites ofchemical alteration along the hybrid polypeptide.

Examples of the hybrid polypeptides of the present invention areprovided in the Sequence Listing and further discussed in the Examplessection herein.

Use of Hybrid Polypeptides in the Treatment or Prevention of MetabolicConditions or Disorders

Hybrids of the invention can be useful for reducing food intake,reducing appetite, reducing caloric intake, inducing satiety, reducingnutrient availability, causing weight loss, affecting body composition,altering body energy content or energy expenditure, improving lipidprofile (including reducing LDL cholesterol and triglyceride levelsand/or changing HDL cholesterol levels), slowing gastrointestinalmotility, delay gastric emptying, moderating the postprandial bloodglucose excursions, preventing or inhibiting glucagon secretion, anddecreasing blood pressure. In one embodiment such hybrids contain anexendin, GLP1, amylin and/or sCT portion.

Thus, in certain embodiments, the hybrids of the invention are usefulfor treating or preventing conditions or disorders which can bealleviated by reducing nutrient availability comprising administering tosaid subject a therapeutically or prophylactically effective amount of acompound of the invention. Such conditions and disorders include, butare not limited to, eating disorders, insulin-resistance, obesity,abnormal postprandial hyperglycemia, diabetes of any kind, includingType I, Type II, and gestational diabetes, Metabolic Syndrome, DumpingSyndrome, hypertension, dyslipidemia, cardiovascular disease,hyperlipidemia, sleep apnea, cancer, pulmonary hypertension,cholecystitis, and osteoarthritis. In one embodiment such hybridscontain an exendin, GLP1, amylin and/or sCT portion.

Exemplary peptide module pairings include cardioactive/protectivepeptides, for example a urocortin with a GLP-1 or exendin, an ANP, BNPor CNP with a GLP-1 or exendin, and a urocortin with an ANP, BNP or CNPSuch hybrids will be cardioprotective and particularly useful for therelated diseases and conditions described herein, including acute orchronic CHF, ischemia reperfusion, myocardial infarction, and forvasodilator actions useful to treat or prevent antihypertensiveindications and angina. Ucn 2 and 3 are particularly useful in hybridsof the invention.

Non-limiting examples of a cardiovascular condition or disease arehypertension, myocardial ischemia, and myocardial reperfusion. Compoundsof the invention may also be useful in treating or preventing otherconditions associated with obesity including stroke, cancer (e.g.,endometrial, breast, prostate, and colon cancer), gallbladder disease,sleep apnea, reduced fertility, and osteoarthritis, (see Lyznicki et al,Am. Fam. Phys. 63:2185, 2001). In other embodiments, compounds of theinvention may be used to alter body composition for aesthetic reasons,to enhance one's physical capabilities, or to produce a leaner meatsource. Hybrids are useful to change body composition by decreasing fatwithout significant decrease in muscle mass, thus producing a desirableloss of body fat while preserving lean body mass. In one embodiment suchhybrids contain an exendin, GLP1, amylin and/or sCT portion.

In another general aspect, hybrids of the invention may be used toinhibit the secretion of ghrelin. Accordingly, compounds of theinvention may be utilize this mechanism to treat or prevent ghrelinrelated disorders such as Prader-Willi syndrome, diabetes of all typesand its complications, obesity, hyperphagia, hyperlipidemia, or otherdisorders associated with hypernutrition. In one embodiment such hybridscontain an exendin, GLP1, amylin and/or sCT portion.

In another general aspect, it is now recognized that hybrids containingamylin and/or sCT portions can be useful for treating or preventingBarrett's esophagus, Gastroesophageal Reflux Disease (GERD) andconditions associated therewith. Such conditions can include, but arenot limited to, heartburn, heartburn accompanied by regurgitation ofgastric/intestinal contents into the mouth or the lungs, difficulty inswallowing, coughing, intermittent wheezing and vocal cord inflammation(conditions associated with GERD), esophageal erosion, esophageal ulcer,esophageal stricture, Barrett's metaplasia (replacement of normalesophageal epithelium with abnormal epithelium), Barrett'sadenocarcinoma, and pulmonary aspiration. Such hybrids haveanti-secretory properties, such as inhibition of gastric acids,inhibition of bile acids, and inhibition of pancreatic enzymes.Moreover, such hybrids can have a gastroprotective effect, which rendersthem particularly useful in the treatment or prevention of Barrett'sesophagus, and/or GERD and related or associated conditions as describedherein.

In another general aspect, hybrids can be further be useful for treatingor preventing pancreatitis, pancreatic carcinoma, and gastritis,particularly in the treatment and prevention of pancreatitis in patientswho have undergone endoscopic retrograde cholangiopancreatography(ERCP). Amylin and/or sCT containing hybrid agonists can have asuprisingly superior therapeutic effect when combined with somatostatin.Accordingly, in certain embodiments, methods for treating or preventingpancreatitis comprise administering such hybrids and administeringsomatostatin and somatostatin agonists to a subject.

In another general aspect, hybrids are useful for decreasing boneresorption, decreasing plasma calcium, and inducing an analgesic effect,particularly to treat bone disorders such as osteopenia andosteoporosis. In yet other embodiments, hybrids are useful to treat painand painful neuropathy. In one embodiment such hybrids contain anexendin, GLP1, amylin and/or sCT portion.

In another aspect of the invention, methods for treating or preventingobesity are provided, wherein the method comprises administering atherapeutically or prophylactically effective amount of a hybridpolypeptide to a subject in need thereof. In a preferred embodiment, thesubject is an obese or overweight subject. While “obesity” is generallydefined as a body mass index over 30, for purposes of this disclosure,any subject, including those with a body mass index of less than 30, whoneeds or wishes to reduce body weight is included in the scope of“obese.” Subjects who are insulin resistant, glucose intolerant, or haveany form of diabetes mellitus (e.g., type 1, 2 or gestational diabetes)can benefit from these hybrids. In one embodiment such hybrids containan exendin, PYY, GLP1, amylin and/or sCT portion.

In other aspects of the invention, methods of reducing food intake,reducing nutrient availability, causing weight loss, affecting bodycomposition, and altering body energy content or increasing energyexpenditure, treating diabetes mellitus, and improving lipid profile(including reducing LDL cholesterol and triglyceride levels and/orchanging HDL cholesterol levels) are provided, wherein the methodscomprise administering to a subject an effective amount of a hybridpolypeptide of the invention. In a preferred embodiment, the methods ofthe invention are used to treat or prevent conditions or disorders whichcan be alleviated by reducing nutrient availability in a subject in needthereof, comprising administering to said subject a therapeutically orprophylactically effective amount of a hybrid polypeptide of theinvention. Such conditions and disorders include, but are not limitedto, hypertension, dyslipidemia, cardiovascular disease, eatingdisorders, insulin-resistance, obesity, and diabetes mellitus of anykind. In one embodiment such hybrids contain an exendin, PYY, GLP1,amylin and/or sCT portion.

Without intending to be limited by theory, it is believed that theeffects of peripherally-administered hybrid polypeptides of the presentinvention in the reduction of food intake, in the delay of gastricemptying, in the reduction of nutrient availability, and in thecausation of weight loss are determined by interactions with one or moreunique receptor classes in, or similar to, those in the PP family. Moreparticularly, it appears that a receptor or receptors similar to thePYY-preferring (or Y7) receptors are involved.

Additional assays useful to the invention include those that candetermine the effect of PPF compounds on body composition. An exemplaryassay can be one that involves utilization of a diet-induced obese (DIO)mouse model for metabolic disease. Prior to the treatment period, maleC57BL/6J mice can be fed a high-fat diet (#D12331, 58% of calories fromfat; Research Diets, Inc.,) for 6 weeks beginning at 4 weeks of age.During the study, the mice can continue to eat their high-fat diet.Water can be provided ad libitum throughout the study. One group ofsimilarly-aged non-obese mice can be fed a low-fat diet (#D12329, 11% ofcalories from fat) for purposes of comparing metabolic parameters to DIOgroups.

DIO mice can be implanted with subcutaneous (SC) intrascapular osmoticpumps to deliver either vehicle (50% dimethylsulfoxide (DMSO) in water)or a compound of the invention. The pumps of the latter group can be setto deliver any amount, e.g., 1000 μg/kg/d of a compound of the inventionfor 7-28 days.

Body weights and food intake can be measured over regular intervalsthroughout the study periods. Respiratory quotient (RQ, defined as CO₂production÷O₂ consumption) and metabolic rate can be determined usingwhole-animal indirect calorimetry (Oxymax, Columbus Instruments,Columbus, Ohio). The mice can be euthanized by isoflurane overdose, andan index of adiposity (bilateral epididymal fat pad weight) measured.Moreover, prior to determination of epididymal weight, body composition(lean mass, fat mass) for each mouse can be analyzed using a Dual EnergyX-ray Absorptiometry (DEXA) instrument per manufacturer's instructions(Lunar Piximus, GE Imaging System). In the methods of the invention,preferred PPF polypeptide of the invention are those having a potency inone of the assays described herein (preferably food intake, gastricemptying, pancreatic secretion, weight reduction or body compositionassays) which is greater than the potency of a component peptide hormonein that same assay.

In addition to the amelioration of hypertension in subjects in needthereof as a result of reduced food intake, weight loss, or treatingobesity, compounds of the invention may be used to treat hypotension.

Compounds of the invention may also be useful for potentiating,inducing, enhancing or restoring glucose responsivity in pancreaticislets or cells. These actions may be useful for treating or preventingconditions associated with metabolic disorders such as those describedabove and in U.S. patent application no. US20040228846. Assays fordetermining such activity are known in the art. For example, inpublished U.S. patent application no. US20040228846 (incorporated byreference in its entirety), assays are described for islet isolation andculture as well as determining fetal islet maturation. In the examplesof patent application US20040228846, intestine-derived hormone peptidesincluding pancreatic polypeptide (PP), neuropeptide Y (NPY),neuropeptide K (NPK), PYY, secretin, glucagon-like peptide-1 (GLP-1) andbombesin were purchased from Sigma. Collagenase type XI was obtainedfrom Sigma. RPMI 1640 culture medium and fetal bovine serum wereobtained from Gibco. A radioimmunoassay kit containing anti-insulinantibody ([¹²⁵I]-RIA kit) was purchased from Linco, St Louis.

Post-partem rat islets were obtained from P-02 year old rats. Adult ratislets were obtained from 6-8 week old rats. Fetal rat islets wereobtained as follows. Pregnant female rats were sacrificed on pregnancyday E21. Fetuses were removed from the uterus. 10-14 pancreata weredissected from each litter and washed twice in Hanks buffer. Thepancreata were pooled, suspended in 6 ml 1 mg/ml collagenase (Type XI,Sigma) and incubated at 37° C. for 8-10 minutes with constant shaking.The digestion was stopped by adding 10 volumes of ice-cold Hanks bufferfollowed by three washes with Hanks buffer. The islets were thenpurified by Ficoll gradient and cultured in 10% fetal bovine serum(FBS)/RPMI medium with or without addition of 1 μM IBMX. At the end offive days, 20 islets were hand picked into each tube and assayed forstatic insulin release. Generally, islets were first washed with KRPbuffer and then incubated with 1 ml of KRP buffer containing 3 mM (low)glucose for 30 minutes at 37° C. with constant shaking After collectingthe supernatant, the islets were then incubated with 17 mM (high)glucose for one hour at 37° C. The insulin released from low or highglucose stimulation were assayed by radioimmunoassay (RIA) using the[¹²⁵I]-RIA kit. E21 fetal islets were cultured for 5 days in thepresence of 200 ng/ml PYY, PP, CCK, NPK, NPY, Secretin, GLP-1 orBombesin.

An exemplary in vivo assay is also provided using the Zucker DiabeticFatty (ZDF) male rat, an inbred (>F30 Generations) rat model thatspontaneously expresses diabetes in all fa/fa males fed a standardrodent diet Purina 5008. In ZDF fa-fa males, hyperglycemia begins todevelop at about seven weeks of age and glucose levels (fed) typicallyreach 500 mg/DL by 10 to 11 weeks of age. Insulin levels (fed) are highduring the development of diabetes. However, by 19 weeks of age insulindrops to about the level of lean control litter mates. Triglyceride andcholesterol levels of obese rats are normally higher than those ofleans. In the assay, three groups of 7-week old ZDF rats, with 6 ratsper group, received the infusion treatment by ALZA pump for 14 days: 1)vehicle control, 2) and 3), PYY with two different doses, 100 pmol/kg/hrand 500 pmol/kg/hr respectively. Four measurements were taken before theinfusion and after the infusion at day 7 and day 14: 1) plasma glucoselevel, 2) plasma insulin level, and 3) plasma triglycerides (TG) level,as well as oral glucose tolerance (OGTT) test. Accordingly, these assayscan be used with compounds of the invention to test for desiredactivity.

Other uses contemplated for the hybrid polypeptides include methods forreducing aluminum (Al) concentrations in the central nervous system (seeU.S. Pat. No. 6,734,166, incorporated by reference in its entirety) fortreating, preventing, or delay the onset of Alzheimer's disease. Assaysfor determining effects on Al are known in the art and can be found inU.S. Pat. No. 6,734,166 using diploid and Ts mice. These mice wereindividually housed in Nalgene® brand metabolism or polypropylene cagesand given three days to adjust to the cages before experimentation. Micehad free access to food (LabDiet® NIH Rat and Moust/Auto 6F5K52, St.Louis, Mo.) and water during the experiment except for the 16 hoursprior to euthanasia when no food was provided. Mice were given dailysubcutaneous injections of either active compound or saline. Mice weresacrificed at the end of day 13 for one experiment and day 3 foranother, and samples were collected. Mice brain samples were weighted inclean teflon liners and prepared for analysis by microwave digestion inlow trace element grade nitric acid. Samples were then analyzed for Alcontent using Inductively Coupled Plasma Mass Spectrometry (Nuttall etal., Annals of Clinical and Laboratory Science 25, 3, 264-271 (1995)).All tissue handling during analysis took place in a clean roomenvironment utilizing HEPA air filtration systems to minimize backgroundcontamination. Hybrids of the invention are useful for prevention andtreatment of nephropathy, including hypertensive and diabeticnephropathy, and nephropathy associated with insulin resistance andmetabolic syndrome. Hybrids achieve these ends by, among other things,improving or preventing worsening of hypertension, endothelial function,renal function, and glomerulosclerosis. In one embodiment, the inventionprovides a method for preventing or treating nephropathy, includinghypertensive and diabetic nephropathy, or that related to insulinresistance, comprising administering a compound of the invention.Hybrids find further use for improving endothelial function in a patienthaving reduced vasodilatory capacity, or having glomerulosclerosis orany other reduction in glomerular flow. Such improvement in endothelialfunction serves both to reduce hypertension and to improve the functionof the capillaries of the glomeruli. In additional embodiments, themolecules of the invention are useful to prevent progression ofnephropathy to ESRD, to prevent, slow the progression of, treat orameliorate proteinuria and/or glomerulosclerosis. Hybrids are useful forreducing the risk of suffering from, preventing, or treating cardiacarrhythmias. Hybrids can provide anti-arrhythmic effects in patientswith cardiac ischemia, cardiac ischemia-reperfusion, and congestiveheart failure. For example, GLP-1 has been found to reduce cardiacinjury and enhance recovery in patients with these disorders. Incretins,including GLP-1, are glucose-dependent insulinotropic hormones. GLP-1and exendin effectively enhance peripheral glucose uptake withoutinducing dangerous hypoglycemia. They also strongly suppress glucagonsecretion, independent of its insulinotropic action, and therebypowerfully reduce plasma free fatty acid (FFA) levels substantially morethan can be accomplished with insulin. High FFA levels have beenimplicated as a major toxic mechanism during myocardial ischemia. Inanother embodiment hybrids are useful for preventing and treatingcardiac arrhythmias that reliably reduce injury associated withreperfusion and ischemia, and enhance patient recovery. In yet a furtherembodiment hybrid treatment after acute stroke or hemorrhage, preferablyintravenous administration, provides a means for optimizing insulinsecretion, increasing brain anabolism, enhancing insulin effectivenessby suppressing glucagon, and maintaining euglycemia or mild hypoglycemiawith no risk of severe hypoglycemia or other adverse side effects. Inone embodiment such hybrids contain a GLP1 or exendin portion. In afurther embodiment a GLP1 or exendin family module is combined with anatriuretic family peptide, an amylin family peptide, a urocortin familypeptide module to obtain enhanced treatment or prevention ofcardiovascular conditons or diseases, including CHF, as describedherein.

In yet a further embodiment hybrids that are capable of lowering insulinresistance or increasing insulin sensitivity are useful to treatpolycystic ovary syndrome (PCOS). Administering hybrids of the inventioncan reduce or prevent insulin resistance in a subject suffering fromPCOS. In yet another embodiment hybrids prevent the onset of type-2diabetes in a subject suffering from PCOS. Further hybrids can restoreregular menses, ovulation, or fertility in a subject suffering fromPCOS. In one embodiment such hybrids contain a GLP1 or an exendinportion for binding and activating a GLP 1 receptor.

The compounds of the invention exhibit a broad range of biologicalactivities, some related to their antisecretory and antimotilityproperties. The compounds may suppress gastrointestinal secretions bydirect interaction with epithelial cells or, perhaps, by inhibitingsecretion of hormones or neurotransmitters which stimulate intestinalsecretion. Anti-secretory properties include inhibition of gastricand/or pancreatic secretions and can be useful in the treatment orprevention of diseases and disorders including gastritis, pancreatitis,Barrett's esophagus, and Gastroesophageal Reflux Disease.

Compounds of the invention are useful in the treatment of any number ofgastrointestinal disorders (see e.g., Harrison's Principles of InternalMedicine, McGraw-Hill Inco, New York, 12th Ed.) that are associated withexcess intestinal electrolyte and water secretion as well as decreasedabsorption, e.g., infectious diarrhea, inflammatory diarrhea, shortbowel syndrome, or the diarrhea which typically occurs followingsurgical procedures, e.g., ileostomy. Examples of infectious diarrheainclude, without limitation, acute viral diarrhea, acute bacterialdiarrhea (e.g., salmonella, campylobacter, and clostridium or due toprotozoal infections), or traveller's diarrhea (e.g., Norwalk virus orrotavirus). Examples of inflammatory diarrhea include, withoutlimitation, malabsorption syndrome, tropical sprue, chronicpancreatitis, Crohn's disease, diarrhea, and irritable bowel syndrome.It has also been discovered that the peptides of the invention can beused to treat an emergency or life-threatening situation involving agastrointestinal disorder, e.g., after surgery or due to cholera.

Compounds of the invention may also be useful for treating or preventingintestinal damage as opposed to merely treating the symptoms associatedwith the intestinal damage (for example, diarrhea). Such damage to theintestine may be, or a result of, ulcerative colitis, inflammatory boweldisease, bowel atrophy, loss bowel mucosa, and/or loss of bowel mucosalfunction (see WO 03/105763, incorporated herein by reference in itsentirety). Assays for such activity, as described in WO 03/105763,include 11 week old male HSD rats, ranging 250-300 grams housed in a12:12 light:dark cycle, and allowed ad libitum access to a standardrodent diet (Teklad LM 485, Madison, Wis.) and water. The animals werefasted for 24 hours before the experiment. A simple and reproducible ratmodel of chronic colonic inflammation has been previously described byMorris G P, et al., “Hapten-induced model of chronic inflammation andulceration in the rat colon.” Gastroenterology. 1989; 96:795-803. Itexhibits a relatively long duration of inflammation and ulceration,affording an opportunity to study the pathophysiology of colonicinflammatory disease in a specifically controlled fashion, and toevaluate new treatments potentially applicable to inflammatory boweldisease in humans.

Rats were anesthetized with 3% isofluorane and placed on a regulatedheating pad set at 37° C. A gavage needle was inserted rectally into thecolon 7 cm. The hapten trinitrobenzenesulfonic acid (TNBS) dissolved in50% ethanol (v/v) was delivered into the lumen of the colon through thegavage needle at a dose of 30 mg/kg, in a total volume of 0 0.4-0.6 mL,as described in Mazelin, et al., Juton Nery Syst. 1998; 73:38 45.Control groups received saline solution (NaCl 0.9%) intracolonically.

Four days after induction of colitis, the colon was resected fromanesthetized rats, which were then euthanized by decapitation. Weightsof excised colon and spleen were measured, and the colons photographedfor scoring of gross morphologic damage. Inflammation was defined asregions of hyperemia and bowel wall thickening.

Hybrid polypeptides of the invention may also be used to treat orprevent pancreatic tumors (e.g., inhibit the proliferation of pancreatictumors). Methods of the invention include reducing the proliferation oftumor cells. The types of benign pancreatic tumor cells which may betreated in accordance with the present invention include serous cystadenomas, microcystic tumors, and solid-cystic tumors. The method isalso effective in reducing the proliferation of malignant pancreatictumor cells such as carcinomas arising from the ducts, acini, or isletsof the pancreas. U.S. Pat. No. 5,574,010 (incorporated by reference inits entirety) provides exemplary assays for testing anti-proliferativeproperties. For example, the '010 patent provides that PANC-1 andMiaPaCa-2 are two human pancreatic adenocarcinoma cancer cell lineswhich are available commercially from suppliers such as American TypeCulture Collection, ATCC (Rockville, Md.). The two tumor cells weregrown in RPMI-1640 culture media supplemented with 10% fetal bovineserum, 29.2 mg/L of glutamine, 25 μg gentamicin, 5 ml penicillin,streptomycin, and fungizone solution (JRH Biosciences, Lenexa, Kans.) at37 degrees Celcius in a NAPCO water jacketed 5% CO₂ incubator. All celllines were detached with 0.25% trypsin (Clonetics, San Diego, Calif.)once to twice a week when a confluent monolayer of tumor cells wasachieved. Cells were pelleted for 7 minutes at 500 g in a refrigeratedcentrifuge at 4 degrees Celcius, and resuspended in trypsin freefortified RPMI 1640 culture media. Viable cells were counted on ahemocytometer slide with trypan blue.

Ten thousand, 20,000, 40,000 and 80,000 cells of each type were added to96 well microculture plates (Costar, Cambridge, Mass.) in a total volumeof 200 ul of culture media per well. Cells were allowed to adhere for 24hours prior to addition of the PYY or test peptide. Fresh culture mediawas exchanged prior to addition of peptides. In vitro incubation ofpancreatic tumor cells with either PYY or test compound was continuedfor 6 hours and 36 hours in length. PYY was added to cells at doses of250 pmol, 25 pmol, and 2.5 pmol per well (N=14). Test compound was addedto cells cultures at doses of 400 pmol, 40 pmol, and 4 pmol per well.Control wells received 2 ul of 0.9% saline to mimic the volume andphysical disturbance upon adhered tumor cells. Each 96 well platecontained 18 control wells to allow for comparison within each plateduring experimentation. Ninety-six (96) well plates were repeated 6times with varying concentrations of PYY and test compound in both thePANC-1 and MiaPaCa-2 cells.

At the end of the incubation period,3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide, MTrtetrazolium bromide (Sigma, St. Louis, Mo.) was added to fresh culturemedia at 0.5 mg/ml. Culture media was exchanged and tumor cells wereincubated for 4 hours with MTT tetrazolium bromide at 37° C. At the endof incubation, culture media was aspirated. Formazon crystalprecipitates were dissolved in 200 μl of dimethyl sulfoxide (Sigma, St.Louis, Mo.). Quantitation of solubilized formazon was performed byobtaining absorption readings at 500 nm wavelength on an ELISA reader(Molecular Devices, Menlo Park, Calif.). The MTT assay measuresmitochondrial NADH dependent dehydrogenase activity, and it has beenamong the most sensitive and reliable method to quantitative in vitrochemotherapy responses of tumor cells. (Alley, M. C., et al., CancerRes., 48:589-601, 1988; Carmichael, J., et al., Cancer Res., 47:936-942,1987; McHale, A. P., et al., Cancer Lett., 41:315-321, 1988; and Saxton,R. E., et al., J. Clin. Laser Med. and Surg., 10(5):331-336, 1992.)Analysis of absorption readings at 550 nm were analyzed by groupingwells of the same test conditions and verifying differences occurringbetween control and the various peptide concentration treatments byone-way ANOVA.

An exemplary in vivo assay is also provided. The human pancreatic ductaladenocarcinoma Mia Paca-2 was examined for in vivo growth inhibition bypeptide YY and test compound. Seventy thousand to 100,000 human MiaPaCa-2 cells were orthotopically transplanted into 48 male athymic mice.After one week, the animals were treated with either PYY or testcompound at 200 pmol/kg/hr via mini-osmotic pumps for four weeks. Thepaired cultures received saline. At sacrifice, both tumor size and masswere measured. Control mice had significant human cancer growth withinthe pancreas as evidenced by histologic sections. At 9 weeks, ninetypercent (90%) of control mice had substantial metastatic disease. Tumormass was decreased by 60.5% in test treated mice and 27% in PYY treatedmice.

Hybrids are also useful for the therapeutic and prophylactic treatmentof neurological and nervous system disorders associated with neuronalloss or dysfunction, including, but not limited to, Parkinson's Disease,Alzheimer's Disease, Huntington's Disease, ALS, stroke, ADD, andneuropsychiatric syndromes, and to enhance or facilitate learning,memory and cognition in mammals. Particularly useful in this regard arehybrids containing an exendin or GLP1 active portion, more specificallycomprising at least the N-terminal 7-15 amino acids or analog thereof,for example HSEGTFTSD (SEQ ID NO:378).

For all indications, in preferred embodiments, the hybrid polypeptide ofthe invention is administered peripherally at a dose of about 0.5 μg toabout 5 mg per day in single or divided doses or controlled continualrelease, or at about 0.01 μg/kg to about 500 μg/kg per dose, morepreferably about 0.05 μg/kg to about 250 μg/kg, most preferably belowabout 50 μg/kg. Dosages in these ranges will vary with the potency ofeach analog or derivative, of course, and may be determined by one ofskill in the art.

In the methods of the present invention, hybrid polypeptides of theinvention may be administered separately or together with one or moreother compounds and compositions that exhibit a long term or short-termaction to reduce nutrient availability, including, but not limited toother compounds and compositions that comprise an amylin or amylinanalog agonist, salmon calcitonin, a cholecystokinin (CCK) or CCKagonist, a leptin (OB protein) or leptin agonist, an exendin or exendinanalog agonist, or a GLP-1 or GLP-1 analog agonist. Suitable amylinagonists include, for example, [^(25,28,29)Pro-] human amylin (SEQ IDNO:67) (also known as “pramlintide,” and described in U.S. Pat. Nos.5,686,511 and 5,998,367). The CCK used is preferably CCK octapeptide(CCK-8), more preferably its sulfated form. Leptin is discussed in, forexample, (Pelleymounter et al., Science 269: 540-3 (1995); Halaas etal., Science 269: 543-6 (1995); Campfield et al., Science 269: 546-9(1995)). Suitable exendins include exendin-3 and exendin-4, and exendinagonist compounds include, for example, those described in PCTPublications WO 99/07404, WO 99/25727, and WO 99/25728.

Polypeptide Production and Purification

The hybrid polypeptides described herein may be prepared using standardrecombinant techniques or chemical peptide synthesis techniques known inthe art, e.g., using an automated or semi-automated peptide synthesizer,or both.

The hybrid polypeptides of the invention can be synthesized in solutionor on a solid support in accordance with conventional techniques.Various automatic synthesizers are commercially available and can beused in accordance with known protocols. See, e.g., Stewart and Young,Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co. (1984); Tamet al., J. Am. Chem. Soc. 105: 6442 (1983); Merrifield, Science 232:341-7 (1986); and Barany and Merrifield, The Peptides, Gross andMeienhofer, eds., Academic Press, New York, 1-284 (1979). Solid phasepeptide synthesis may be carried out with an automatic peptidesynthesizer (e.g., Model 430A, Applied Biosystems Inc., Foster City,Calif.) using the NMP/HOBt (Option 1) system and tBoc or Fmoc chemistry(see, Applied Biosystems User's Manual for the ABI 430A PeptideSynthesizer, Version 1.3B Jul. 1, 1988, section 6, pp. 49-70, AppliedBiosystems, Inc., Foster City, Calif.) with capping. Peptides may alsobe assembled using an Advanced Chem Tech Synthesizer (Model MPS 350,Louisville, Ky.). Peptides may be purified by RP-HPLC (preparative andanalytical) using, e.g., a Waters Delta Prep 3000 system and a C4, C8,or C18 preparative column (10μ, 2.2×25 cm; Vydac, Hesperia, Calif.). Theactive peptide can be readily synthesized and then screened in screeningassays designed to identify reactive peptides.

The hybrid polypeptides of the present invention may alternatively beproduced by recombinant techniques well known in the art. See, e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., ColdSpring Harbor (1989). These hybrid polypeptides produced by recombinanttechnologies may be expressed from a polynucleotide. One skilled in theart will appreciate that the polynucleotides, including DNA and RNA,that encode such the various fragments of the hybrid polypeptides may beobtained from the wild-type cDNA, taking into consideration thedegeneracy of codon usage, or may be engineered as desired. Thesepolynucleotide sequences may incorporate codons facilitatingtranscription and translation of mRNA in microbial hosts. Suchmanufacturing sequences may readily be constructed according to themethods well known in the art. See, e.g., WO 83/04053. Thepolynucleotides above may also optionally encode an N-terminal methionylresidue. Non-peptide compounds useful in the present invention may beprepared by art-known methods. For example, phosphate-containing aminoacids and peptides containing such amino acids may be prepared usingmethods known in the art. See, e.g., Bartlett and Landen, Bioorg. Chem.14: 356-77 (1986).

A variety of expression vector/host systems may be utilized to containand express a hybrid polypeptide coding sequence. These include but arenot limited to microorganisms such as bacteria transformed withrecombinant bacteriophage, plasmid or cosmid DNA expression vectors;yeast transformed with yeast expression vectors; insect cell systemsinfected with virus expression vectors (e.g., baculovirus); plant cellsystems transfected with virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withbacterial expression vectors (e.g., Ti or pBR322 plasmid); or animalcell systems. Mammalian cells that are useful in recombinant proteinproductions include but are not limited to VERO cells, HeLa cells,Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), WI38, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293 cells.Exemplary protocols for the recombinant expression of the protein aredescribed herein.

As such, polynucleotide sequences provided by the invention are usefulin generating new and useful viral and plasmid DNA vectors, new anduseful transformed and transfected procaryotic and eucaryotic host cells(including bacterial, yeast, and mammalian cells grown in culture), andnew and useful methods for cultured growth of such host cells capable ofexpression of the present hybrid polypeptides. The polynucleotidesequences encoding hybrid polypeptides herein may be useful for genetherapy in instances where underproduction of the component peptidehormone(s) of the chimera would be alleviated, or the need for increasedlevels of such would be met.

The present invention also provides for processes for recombinant DNAproduction of the present hybrid polypeptides. Provided is a process forproducing the hybrid polypeptides from a host cell containing nucleicacids encoding such hybrid polypeptides comprising: (a) culturing saidhost cell containing polynucleotides encoding such hybrid polypeptidesunder conditions facilitating the expression of such DNA molecule; and(b) obtaining such hybrid polypeptides.

Host cells may be prokaryotic or eukaryotic and include bacteria,mammalian cells (such as Chinese Hamster Ovary (CHO) cells, monkeycells, baby hamster kidney cells, cancer cells or other cells), yeastcells, and insect cells.

Mammalian host systems for the expression of the recombinant proteinalso are well known to those of skill in the art. Host cell strains maybe chosen for a particular ability to process the expressed protein orproduce certain post-translation modifications that will be useful inproviding protein activity. Such modifications of the polypeptideinclude, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing, which cleaves a “prepro” form of theprotein, may also be important for correct insertion, folding and/orfunction. Different host cells, such as CHO, HeLa, MDCK, 293, W138, andthe like, have specific cellular machinery and characteristic mechanismsfor such post-translational activities, and may be chosen to ensure thecorrect modification and processing of the introduced foreign protein.

Alternatively, a yeast system may be employed to generate the hybridpolypeptides of the present invention. The coding region of the hybridpolypeptide cDNA is amplified by PCR. A DNA encoding the yeastpre-pro-alpha leader sequence is amplified from yeast genomic DNA in aPCR reaction using one primer containing nucleotides 1-20 of the alphamating factor gene and another primer complementary to nucleotides255-235 of this gene (Kurjan and Herskowitz, Cell, 30: 933-43 (1982)).The pre-pro-alpha leader coding sequence and hybrid polypeptide codingsequence fragments are ligated into a plasmid containing the yeastalcohol dehydrogenase (ADH2) promoter, such that the promoter directsexpression of a fusion protein consisting of the pre-pro-alpha factorfused to the mature hybrid polypeptide. As taught by Rose and Broach,Meth. Enz. 185: 234-79, Goeddel ed., Academic Press, Inc., San Diego,Calif. (1990), the vector further includes an ADH2 transcriptionterminator downstream of the cloning site, the yeast “2-micron”replication origin, the yeast leu-2d gene, the yeast REP1 and REP2genes, the E. coli β-lactamase gene, and an E. coli origin ofreplication. The β-lactamase and leu-2d genes provide for selection inbacteria and yeast, respectively. The leu-2d gene also facilitatesincreased copy number of the plasmid in yeast to induce higher levels ofexpression. The REP1 and REP2 genes encode proteins involved inregulation of the plasmid copy number.

The DNA construct described in the preceding paragraph is transformedinto yeast cells using a known method, e.g., lithium acetate treatment(Steams et al., Meth. Enz. 185: 280-97 (1990)). The ADH2 promoter isinduced upon exhaustion of glucose in the growth media (Price et al.,Gene 55: 287 (1987)). The pre-pro-alpha sequence effects secretion ofthe fusion protein from the cells. Concomitantly, the yeast KEX2 proteincleaves the pre-pro sequence from the mature PYY analog polypeptides(Bitter et al., Proc. Natl. Acad. Sci. USA 81: 5330-4 (1984)).

Hybrid polypeptides of the invention may also be recombinantly expressedin yeast using a commercially available expression system, e.g., thePichia Expression System (Invitrogen, San Diego, Calif.), following themanufacturer's instructions. This system also relies on thepre-pro-alpha sequence to direct secretion, but transcription of theinsert is driven by the alcohol oxidase (AOX1) promoter upon inductionby methanol. The secreted hybrid polypeptide is purified from the yeastgrowth medium by, e.g., the methods used to purify hybrid polypeptidefrom bacterial and mammalian cell supernatants.

Alternatively, the cDNA encoding hybrid polypeptides may be cloned intothe baculovirus expression vector pVL1393 (PharMingen, San Diego,Calif.). This hybrid polypeptide-containing vector is then usedaccording to the manufacturer's directions (PharMingen) to infectSpodoptera frugiperda cells in sF9 protein-free media and to producerecombinant protein. The protein is purified and concentrated from themedia using a heparin-Sepharose column (Pharmacia, Piscataway, N.J.) andsequential molecular sizing columns (Amicon, Beverly, Mass.), andresuspended in PBS. SDS-PAGE analysis shows a single band and confirmsthe size of the protein, and Edman sequencing on a Proton 2090 PeptideSequencer confirms its N-terminal sequence.

For example, the DNA sequence encoding the hybrid polypeptide may becloned into a plasmid containing a desired promoter and, optionally, aleader sequence (see, e.g., Better et al., Science 240: 1041-3 (1988)).The sequence of this construct may be confirmed by automated sequencing.The plasmid is then transformed into E. coli, strain MC1061, usingstandard procedures employing CaCl₂ incubation and heat shock treatmentof the bacteria (Sambrook et al., supra). The transformed bacteria aregrown in LB medium supplemented with carbenicillin, and production ofthe expressed protein is induced by growth in a suitable medium. Ifpresent, the leader sequence will affect secretion of the hybridpolypeptide and be cleaved during secretion. The secreted recombinantprotein is purified from the bacterial culture media by the methoddescribed herein.

Alternatively, the hybrid polypeptides of the invention may be expressedin an insect system. Insect systems for protein expression are wellknown to those of skill in the art. In one such system, Autographacalifornica nuclear polyhedrosis virus (AcNPV) is used as a vector toexpress foreign genes in Spodoptera frugiperda cells or in Trichoplusialarvae. The hybrid polypeptide coding sequence is cloned into anonessential region of the virus, such as the polyhedrin gene, andplaced under control of the polyhedrin promoter. Successful insertion ofhybrid polypeptide will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein coat. The recombinant viruses arethen used to infect S. frugiperda cells or Trichoplusia larvae in whichhybrid polypeptide is expressed (Smith et al., J. Virol. 46: 584 (1983);Engelhard et al., Proc. Natl. Acad. Sci. USA 91: 3224-7 (1994)).

In another example, the DNA sequence encoding the hybrid polypeptide maybe amplified by PCR and cloned into an appropriate vector, for example,pGEX-3X (Pharmacia, Piscataway, N.J.). The pGEX vector is designed toproduce a fusion protein comprising glutathione-S-transferase (GST),encoded by the vector, and a protein encoded by a DNA fragment insertedinto the vector's cloning site. The primers for the PCR may be generatedto include, for example, an appropriate cleavage site. The recombinantfusion protein may then be cleaved from the GST portion of the fusionprotein. The pGEX-3X/PYY analog polypeptide construct is transformedinto E. coli XL-1 Blue cells (Stratagene, La Jolla, Calif.), andindividual transformants are isolated and grown at 37° C. in LB medium(supplemented with carbenicillin) to an optical density at wavelength600 nm of 0.4, followed by further incubation for 4 hours in thepresence of 0.5 mM Isopropyl β-D-Thiogalactopyranoside (Sigma ChemicalCo., St. Louis, Mo.). Plasmid DNA from individual transformants ispurified and partially sequenced using an automated sequencer to confirmthe presence of the desired PPF hybrid polypeptide-encoding gene insertin the proper orientation.

The fusion protein, expected to be produced as an insoluble inclusionbody in the bacteria, may be purified as follows. Cells are harvested bycentrifugation; washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA; andtreated with 0.1 mg/mL lysozyme (Sigma Chemical Co.) for 15 min. at roomtemperature. The lysate is cleared by sonication, and cell debris ispelleted by centrifugation for 10 min. at 12,000×g. The fusionprotein-containing pellet is resuspended in 50 mM Tris, pH 8, and 10 mMEDTA, layered over 50% glycerol, and centrifuged for 30 min. at 6000×g.The pellet is resuspended in standard phosphate buffered saline solution(PBS) free of Mg⁺⁺ and Ca⁺⁺. The fusion protein is further purified byfractionating the resuspended pellet in a denaturing SDS polyacrylamidegel (Sambrook et al., supra). The gel is soaked in 0.4 M KCl tovisualize the protein, which is excised and electroeluted in gel-runningbuffer lacking SDS. If the GST/PYY analog polypeptide fusion protein isproduced in bacteria as a soluble protein, it may be purified using theGST Purification Module (Pharmacia Biotech).

The fusion protein may be subjected to digestion to cleave the GST fromthe PPF hybrid polypeptide. The digestion reaction (20-40 μg fusionprotein, 20-30 units human thrombin (4000 U/mg (Sigma) in 0.5 mL PBS) isincubated 16-48 hrs. at room temperature and loaded on a denaturingSDS-PAGE gel to fractionate the reaction products. The gel is soaked in0.4 M KCl to visualize the protein bands. The identity of the proteinband corresponding to the expected molecular weight of the hybridpolypeptide may be confirmed by partial amino acid sequence analysisusing an automated sequencer (Applied Biosystems Model 473A, FosterCity, Calif.).

In a particularly preferred method of recombinant expression of thehybrid polypeptides of the present invention, 293 cells may beco-transfected with plasmids containing the hybrid polypeptide cDNA inthe pCMV vector (5′ CMV promoter, 3′ HGH poly A sequence) and pSV2neo(containing the neo resistance gene) by the calcium phosphate method.Preferably, the vectors should be linearized with Scal prior totransfection. Similarly, an alternative construct using a similar pCMVvector with the neo gene incorporated can be used. Stable cell lines areselected from single cell clones by limiting dilution in growth mediacontaining 0.5 mg/mL G418 (neomycin-like antibiotic) for 10-14 days.Cell lines are screened for hybrid polypeptide expression by ELISA orWestern blot, and high-expressing cell lines are expanded for largescale growth.

It is preferable that the transformed cells are used for long-term,high-yield protein production and as such stable expression isdesirable. Once such cells are transformed with vectors that containselectable markers along with the desired expression cassette, the cellsmay be allowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The selectable marker is designed to conferresistance to selection, and its presence allows growth and recovery ofcells that successfully express the introduced sequences. Resistantclumps of stably transformed cells can be proliferated using tissueculture techniques appropriate to the cell.

A number of selection systems may be used to recover the cells that havebeen transformed for recombinant protein production. Such selectionsystems include, but are not limited to, HSV thymidine kinase,hypoxanthine-guanine phosphoribosyltransferase and adeninephosphoribosyltransferase genes, in tk-, hgprt- or aprt-cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for dhfr, that confers resistance to methotrexate; gpt,that confers resistance to mycophenolic acid; neo, that confersresistance to the aminoglycoside, G418; also, that confers resistance tochlorsulfuron; and hygro, that confers resistance to hygromycin.Additional selectable genes that may be useful include trpB, whichallows cells to utilize indole in place of tryptophan, or hisD, whichallows cells to utilize histinol in place of histidine. Markers thatgive a visual indication for identification of transformants includeanthocyanins, β-glucuronidase and its substrate, GUS, and luciferase andits substrate, luciferin.

Many of the hybrid polypeptides of the present invention may be producedusing a combination of both automated peptide synthesis and recombinanttechniques. For example, a hybrid polypeptide of the present inventionmay contain a combination of modifications including deletion,substitution, and insertion by PEGylation. Such a hybrid polypeptide maybe produced in stages. In the first stage, an intermediate polypeptidecontaining the modifications of deletion, substitution, insertion, andany combination thereof, may be produced by recombinant techniques asdescribed. Then after an optional purification step as described herein,the intermediate polypeptide is PEGylated through chemical modificationwith an appropriate PEGylating reagent (e.g., from Nektar Therapeutics,San Carlos, Calif.) to yield the desired hybrid polypeptide. One skilledin the art will appreciate that the above-described procedure may begeneralized to apply to a hybrid polypeptide containing a combination ofmodifications selected from deletion, substitution, insertion,derivation, and other means of modification well known in the art andcontemplated by the present invention.

It may be desirable to purify the hybrid polypeptides generated by thepresent invention. Peptide purification techniques are well known tothose of skill in the art. These techniques involve, at one level, thecrude fractionation of the cellular milieu to polypeptide andnon-polypeptide fractions. Having separated the polypeptide from otherproteins, the polypeptide of interest may be further purified usingchromatographic and electrophoretic techniques to achieve partial orcomplete purification (or purification to homogeneity). Analyticalmethods particularly suited to the preparation of a pure peptide areion-exchange chromatography, exclusion chromatography, polyacrylamidegel electrophoresis, and isoelectric focusing. A particularly efficientmethod of purifying peptides is reverse phase HPLC, followed bycharacterization of purified product by liquid chromatography/massspectrometry (LC/MS) and Matrix-Assisted Laser Desorption Ionization(MALDI) mass spectrometry. Additional confirmation of purity is obtainedby determining amino acid analysis.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of an encodedprotein or peptide. The term “purified peptide” as used herein, isintended to refer to a composition, isolatable from other components,wherein the peptide is purified to any degree relative to its naturallyobtainable state. A purified peptide therefore also refers to a peptide,free from the environment in which it may naturally occur.

Generally, “purified” will refer to a peptide composition that has beensubjected to fractionation to remove various other components, and whichcomposition substantially retains its expressed biological activity.Where the term “substantially purified” is used, this designation willrefer to a composition in which the peptide forms the major component ofthe composition, such as constituting about 50%, about 60%, about 70%,about 80%, about 90%, about 95% or more of the peptides in thecomposition.

Various techniques suitable for use in peptide purification will be wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies, and the like;heat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

There is no general requirement that the peptides always be provided intheir most purified state. Indeed, it is contemplated that lesssubstantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed, utilizing anHPLC apparatus, will generally result in a greater “-fold” purificationthan the same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

One may optionally purify and isolate such hybrid polypeptides fromother components obtained in the process. Methods for purifying apolypeptide can be found in U.S. Pat. No. 5,849,883. These documentsdescribe specific exemplary methods for the isolation and purificationof G-CSF compositions that may be useful in isolating and purifying thehybrid polypeptides of the present invention. Given the disclosure ofthese patents, it is evident that one of skill in the art would be wellaware of numerous purification techniques that may be used to purifyhybrid polypeptides from a given source.

Also it is contemplated that a combination of anion exchange andimmunoaffinity chromatography may be employed to produce purified hybridpolypeptide compositions of the present invention.

Pharmaceutical Compositions

The present invention also relates to pharmaceutical compositionscomprising a therapeutically or prophylactically effective amount of atleast one hybrid polypeptide of the invention, or a pharmaceuticallyacceptable salt thereof, together with pharmaceutically acceptablediluents, preservatives, solubilizers, emulsifiers, adjuvants and/orcarriers useful in the delivery of the hybrid polypeptides. Suchcompositions may include diluents of various buffer content (e.g.,Tris-HCl, acetate, phosphate), pH and ionic strength; additives such asdetergents and solubilizing agents (e.g., Tween 80, Polysorbate 80),anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives(e.g., thimersol, benzyl alcohol), and bulking substances (e.g.,lactose, mannitol); incorporation of the material into particulatepreparations of polymeric compounds, such as polylactic acid,polyglycolic acid, etc., or in association with liposomes. Suchcompositions will influence the physical state, stability, rate of invivo release, and rate of in vivo clearance of the present hybridpolypeptides. See, e.g., Remington's Pharmaceutical Sciences 1435-712,18th ed., Mack Publishing Co., Easton, Pa. (1990).

In general, the present hybrid polypeptides will be useful in the sameway that the individual component polypeptides are useful in view oftheir pharmacological properties. One preferred use is to peripherallyadminister such hybrid polypeptides for the treatment or prevention ofmetabolic conditions and disorders. In particular, the compounds of theinvention possess activity as agents to reduce nutrient availability,reduce food intake, suppress appetite, and effect weight loss. Inanother embodiment, a preferred use is to administer such hybridpolypeptides for the treatment of diabetes or diabetes relatedconditions and disorders.

The present hybrid polypeptides may be formulated for peripheraladministration, including formulation for injection, oraladministration, nasal administration, pulmonary administration, topicaladministration, or other types of administration as one skilled in theart will recognize. More particularly, administration of thepharmaceutical compositions according to the present invention may bevia any common route so long as the target tissue is available via thatroute. In a preferred embodiment, the pharmaceutical compositions may beintroduced into the subject by any conventional peripheral method, e.g.,by intravenous, intradermal, intramusclar, intramammary,intraperitoneal, intrathecal, retrobulbar, intrapulmonary (e.g., termrelease); by oral, sublingual, nasal, anal, vaginal, or transdermaldelivery, or by surgical implantation at a particular site. Thetreatment may consist of a single dose or a plurality of doses over aperiod of time. Controlled continual release of the compositions of thepresent invention is also contemplated.

The formulation may be liquid or may be solid, such as lyophilized, forreconstitution. Aqueous compositions of the present invention comprisean effective amount of the hybrid polypeptide, dissolved or dispersed ina pharmaceutically acceptable carrier or aqueous medium. The phrase“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce adverse, allergic, orother untoward reactions when administered to an animal or a human. Asused herein, “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in therapeuticcompositions is contemplated. Supplementary active ingredients also canbe incorporated into the compositions. In some cases, it will beconvenient to provide a hybrid polypeptide of the invention and anotherfood-intake-reducing, diabetes treating, plasma glucose-lowering, orplasma lipid-altering agent, such as an amylin, an amylin agonistanalog, a CCK or CCK agonist, or a leptin or leptin agonist, or anexendin or exendin agonist analog, in a single composition or solutionfor administration together. In other cases, it may be more advantageousto administer the additional agent separately from said hybridpolypeptide.

The hybrid polypeptide of the invention may be prepared foradministration as solutions of free base, or pharmacologicallyacceptable salts in water suitably mixed with a surfactant, such ashydroxypropylcellulose. Pharmaceutically-acceptable salts include theacid addition salts (formed with the free amino groups of the protein)and which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups also can be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like. Such products are readily prepared by procedureswell known to those skilled in the art. Dispersions also can be preparedin glycerol, liquid polyethylene glycols, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

In one embodiment, the pharmaceutical compositions of the presentinvention are formulated so as to be suitable for parenteraladministration, e.g., via injection or infusion. Preferably, the hybridpolypeptide is suspended in an aqueous carrier, for example, in anisotonic buffer solution at a pH of about 3.0 to about 8.0, preferablyat a pH of about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 to about 5.0.Useful buffers include sodium citrate-citric acid and sodiumphosphate-phosphoric acid, and sodium acetate/acetic acid buffers. Aform of repository or “depot” slow release preparation may be used sothat therapeutically effective amounts of the preparation are deliveredinto the bloodstream over many hours or days following transdermalinjection or delivery.

The pharmaceutical compositions suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form should be sterile and should befluid to the extent that easy syringability exists. It is also desirablefor the hybrid polypeptide of the invention to be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents (for example, sugars or sodium chloride). Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption (for example, aluminummonostearate and gelatin).

Sterile injectable solutions may be prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle that contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum-drying and freeze-drying techniquesthat yield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof.

Generally, a therapeutically or prophylactically effective amount of thepresent hybrid polypeptides will be determined by the age, weight, andcondition or severity of the diseases, conditions or disorders of therecipient. See, e.g., Remington's Pharmaceutical Sciences 697-773. Seealso Wang and Hanson, Parenteral Formulations of Proteins and Peptides:Stability and Stabilizers, Journal of Parenteral Science and Technology,Technical Report No. 10, Supp. 42:2S (1988). Typically, a dosage ofbetween about 0.001 μg/kg body weight/day to about 1000 μg/kg bodyweight/day, may be used, but more or less, as a skilled practitionerwill recognize, may be used. Dosing may be one or more times daily, orless frequently, and may be in conjunction with other compositions asdescribed herein. It should be noted that the present invention is notlimited to the dosages recited herein.

Appropriate dosages may be ascertained through the use of establishedassays for determining level of metabolic conditions or disorders inconjunction with relevant dose-response data. The final dosage regimenwill be determined by the attending physician, considering factors thatmodify the action of drugs, e.g., the drug's specific activity, severityof the damage and the responsiveness of the patient, the age, condition,body weight, sex and diet of the patient, the severity of any infection,time of administration and other clinical factors. As studies areconducted, further information will emerge regarding appropriate dosagelevels and duration of treatment for specific diseases and conditions.

An effective dose will typically be in the range of about 1 to 30 μg toabout 5 mg/day, preferably about 10 to 30 μg to about 2 mg/day and morepreferably about 5 to 100 μg to about 1 mg/day, most preferably about 5μg to about 500 μg/day, for a 50 kg patient, administered in a single ordivided doses. Preferably, dosages are between about 0.01 to about 100μg/kg/dose. The exact dose to be administered may be determined by oneof skill in the art and is dependent upon the potency of the particularcompound, as well as upon the age, weight and condition of theindividual. Administration should begin whenever, e.g., suppression ofnutrient availability, food intake, weight, blood glucose or plasmalipid modulation is desired, for example, at the first sign of symptomsor shortly after diagnosis of obesity, diabetes mellitus, orinsulin-resistance syndrome. Administration may be by any route, e.g.,injection, preferably subcutaneous or intramuscular, oral, nasal,transdermal, etc. Dosages for certain routes, for example oraladministration, may be increased to account for decreasedbioavailablity, for example, by about 5-100 fold.

Parenteral administration may be carried out with an initial bolusfollowed by continuous infusion to maintain therapeutic circulatinglevels of drug product. Those of ordinary skill in the art will readilyoptimize effective dosages and administration regimens as determined bygood medical practice and the clinical condition of the individualpatient.

The frequency of dosing will depend on the pharmacokinetic parameters ofthe agents and the routes of administration. The optimal pharmaceuticalformulation will be determined by one of skill in the art depending onthe route of administration and the desired dosage. See, e.g.,Remington's Pharmaceutical Sciences, supra, pages 1435-1712. Suchformulations may influence the physical state, stability, rate of invivo release and rate of in vivo clearance of the administered agents.Depending on the route of administration, a suitable dose may becalculated according to body weight, body surface areas or organ size.Further refinement of the calculations necessary to determine theappropriate treatment dose is routinely made by those of ordinary skillin the art without undue experimentation, especially in light of thedosage information and assays disclosed herein, as well as thepharmacokinetic data observed in animals or human clinical trials.

It will be appreciated that the pharmaceutical compositions andtreatment methods of the invention may be useful in fields of humanmedicine and veterinary medicine. Thus the subject to be treated may bea mammal, preferably human or other animal. For veterinary purposes,subjects include for example, farm animals including cows, sheep, pigs,horses and goats, companion animals such as dogs and cats, exotic and/orzoo animals, laboratory animals including mice, rats, rabbits, guineapigs and hamsters; and poultry such as chickens, turkeys, ducks andgeese.

In addition, the present invention contemplates a kit comprising ahybrid polypeptide of the invention, components suitable for preparingsaid hybrid polypeptide of the invention for pharmaceutical application,and instructions for using said hybrid polypeptide and components forpharmaceutical application.

To assist in understanding the present invention, the following examplesare included. The experiments relating to this invention should not, ofcourse, be construed as specifically limiting the invention and suchvariations of the invention, now known or later developed, which wouldbe within the purview of one skilled in the art are considered to fallwithin the scope of the invention as described herein and hereinafterclaimed.

EXAMPLES

The present invention is described in more detail with reference to thefollowing non-limiting examples, which are offered to more fullyillustrate the invention, but are not to be construed as limiting thescope thereof. The examples illustrate the preparation of the presenthybrid polypeptides, and the testing of these hybrid polypeptides of theinvention in vitro and/or in vivo. Those of skill in the art willunderstand that the techniques described in these examples representtechniques described by the inventors to function well in the practiceof the invention, and as such constitute preferred modes for thepractice thereof. However, it should be appreciated that those of skillin the art should in light of the present disclosure, appreciate thatmany changes can be made in the specific methods that are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the invention.

Example 1 Preparation of Hybrid Polypeptides

Peptides of the invention may be assembled on a Symphony peptidesynthesizer (Protein Technologies, Inc.) using Rink amide resin(Novabiochem) with a loading of 0.43-0.49 mmol/g at 0.050-0.100 mmol ora pre-loaded Wang Resin (Fmoc-Tyr(tBu)-Wang resin) 0.63 mmol/g(Novabiochem). Fmoc amino acid (5.0 eq, 0.250-0.500 mmol) residues aredissolved at a concentration of 0.10 M in 1-methyl-2-pyrrolidinone. Allother reagents (HBTU, 1-Hydroxybenzotriazole hydrate andN,N-Diisopropylethylamine) are prepared as 0.55 M Dimethylformamidesolutions. The Fmoc protected amino acids are then coupled to theresin-bound amino acid using, HBTU (2.0 eq, 0.100-0.200 mmol),1-Hydroxybenzotriazole hydrate (1.8 eq, 0.090-0.18 mmol),N,N-Diisopropylethylamine (2.4 eq, 0.120-0.240 mmol) for 2 hours.Following the last amino acid coupling, the peptide is deprotected using20% (v/v) piperidine in dimethylformamide for 1 hour. Once peptidesequence is complete, the Symphony peptide synthesizer is programmed tocleave the resin. Trifluoroacetic acid (TFA) cleavage of the peptidefrom resin is carried out using 93% TFA, 3% phenol, 3% water and 1%triisopropylsilane for 1 hour. The cleaved peptide is precipitated usingtert-butyl methyl ether, pelleted by centrifugation and lyophilized. Thepellet is re-dissolved in water (10-15 mL), filtered and purified viareverse phase HPLC using a C18 column and an acetonitrile/water gradientcontaining 0.1% TFA.

A general procedure for N-capping the peptides of the invention withfatty acids (e.g., octanoic and stearic acids) is as follows: Peptide onrink amide resin (0.1 mmol) is suspended in NMP (5 mL). In a separatevial, HBTU (0.3 mmol), HOBt (0.3 mmol) is dissolved in DMF (5 mL)followed by the addition of DIEA (0.6 mmol). This solution is added tothe resin and this suspension is shaken for 2 hrs. The solvent isfiltered and washed thoroughly with NMP (5 mL×4) and CH₂Cl₂ (20 mL),dried and is subjected to the TFA cleavage for 1 hr. The yield of thedesired peptide is ca. 40 mg after cleavage and purification.

PEG modification may be carried out in solution on a free epsilon-aminogroup of lysine or a terminal amino group of a purified peptide usingcommercially available activated PEG esters. The resulting PEGylatedderivatives are purified to homogeneity by reverse phase HPLC and thepurity is confirmed by LC/MS and MALDI-MS.

Certain exemplary hybrid polypeptides of the invention are shown inTable 1-1. Various modifications to the embodied compounds areenvisioned, such as chemical modifications such as glycosylation, PEGmodifications, etc.; amino acid modifications such as substitutions,insertions and deletions, etc. Further, even though represented asC-terminally amidated, it is understood that the hybrid polypeptides ofthe invention may alternatively be in the free acid form.

TABLE 1-1 Certain Exemplary Hybrid Compounds of the Invention SEQ ID:  1HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-ASLRHYLNLVTRQRY-NH₂  2HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-RHYLNLVTRQRY-NH₂  3HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NRYYASLRHYLNLVTRQRY-NH₂  4HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-βAla-βAla-ASLRHYLNLVTRQRY-NH₂  5HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-βAla-βAla-RHYLNLVTRQRY-NH₂  6HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-βAla-βAla-VTRQRY-NH₂  7HGEGTFTSDLSKQMEEEAVRLFIEWLKN-ASLRHYLNLVTRQRY-NH₂  8HGEGTFTSDLSKQMEEEAVRLFIEWLKN-RHYLNLVTRQRY-NH₂  9HGEGTFTSDLSKQMEEEAVRLFIEWLKN-NRYYASLRHYLNLVTRQRY-NH₂  10HGEGTFTSDLSKQMEEEAVRLFIEWLKN-βAla-βAla-ASLRHYLNLVTRQRY-NH₂  11HGEGTFTSDLSKQMEEEAVRLFIEWLKN-βAla-βAla-RHYLNLVTRQRY-NH₂  12HGEGTFTSDLSKQMEEEAVRLFIEWLKN-βAla-βAla-VTRQRY-NH₂  13HGEGAFTSDLSKQLEEEAVRLFIEFLKNNRYYASLRHYLNLVTRQRY-NH₂  14HGEGAFTSDLSKQLEEEAVRLFIEFLKNASLRHYLNLVTRQRY-NH₂  15HGEGAFTSDLSKQLEEEAVRLFIEFLKNRHYLNLVTRQRY-NH₂  16HGEGAFTSDLSKQLEEENRYYASLRHYLNLVTRQRY-NH₂  17HGEGAFTSDLSKQLEEEAVRLFIEFLKN-βAla-βAla-ASLRHYLNLVTRQRY-NH₂  18HGEGAFTSDLSKQLEEEAVRLFIEFLKN-βAla-βAla-RHYLNLVTRQRY-NH₂  19HGEGAFTSDLSKQLEEEAVRLFIEFLKN-βAla-βAla-VTRQRY-NNH₂  20HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-DY(SO₃)MGWMDF-NH₂  21HGEGTFTSDLSKQMEEEAVRLFIEWLKN-DY(SO₃)MGWMDF-NH₂  22HGEGTFTSDLSKQMEEEAVRLFIEWLKN-DF(CH₂SO₃)MGWMDF-NH₂  23HGEGTFTSDLSKQMEEEAVRLFIEWLKN-[8-amino-3,6-dioxaoctanoyl]-DY(SO₃)MGWMDF-NH₂ 24HGEGTFTSDLSKQMEEEAVRLFIEWLKN-[8-amino-3,6-dioxactoanoyl]-DF(CH₂SO₃)MGWMDF-NH₂ 25 HGEGTFTSDLSKQMEEEAVRLFIEWLKKCNTATCVLGRLSQELHRLQTYPRTNTGSNTY-NH₂  26HGEGTFTSDLSKQMEEEAVRLFIEWLKKANTATAVLG-NH₂  27HGEGTFTSDLSKQMEEEAVRLFIEWLKN-12-Ado-KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY-NH₂ 28HGEGTFTSDLSKQMEEEAVRLFIEWLKN-12-Ado-CNTATCVLGRLSQELHRLQTYPRTNTGSNTY-NH₂ 29HGEGTFTSDLSKQMEEEAVRLFIEWLKN-3,6-dioxaoctanoyl-KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY-NH₂ 30HGEGTFTSDLSKQMEEEAVRLFIEWLKN-3,6-dioxaoctanoyl-CNTATCVLGRLSQELHRLQTYPRTNTGSNTY-NH₂ 31HGEGTFTSDLSKQMEEEAVRLFIEWLKN-5-Apa-KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY-NH₂ 32HGEGTFTSDLSKQMEEEAVRLFIEWLKN-5-Apa-CNTATCVLGRLSQELHRLQTYPRTNTGSNTY-NH₂ 33HGEGTFTSDLSKQMEEEAVRLFIEWLKN-βAla-βAla-KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY-NH₂ 34HGEGTFTSDLSKQMEEEAVRLFIEWLKN-βAla-βAla-CNTATCVLGRLSQELHRLQTYPRTNTGSNTY-NH₂ 35 HGEGTFTSDLSKQMEEEAVRLFIEWLKN-4,7,10-trioxa-13-tridecanaminesuccinimidyl-KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY-NH₂  36HGEGTFTSDLSKQMEEEAVRLFIEWLKN-4,7,10-trioxa-13-tridecanaminesuccinimidyl-CNTATCVLGRLSQELHRLQTYPRTNTGSNTY-NH₂ 203DF(CH2SO3)MGWMDF-GKR-KCNTATCATQRLANELVRLQTYPRTNVGSNTY-NH2  38KCNTATCATQRLANFLVR-RYYASLRHYLNLVTRQRY-NH2  39isocaproyl-STAVL-(Aib)-K(formyl)-LSQEL-(Aib)-K(formyl)-LQT-NRYYASLRHYLNLVTRQRY-NH₂ 40isocaproyl-STAVL-(Aib)-K(formyl)-LSQEL-(Aib)-K(formyl)-L-ELNRYYASLRHYLNLVTRQRY-NH₂ 41

 42

 43

309

310

311

 27 ²⁹12Ado-Exendin(1-28)-hAmylin(1-7)-^(11,18)Arg-sCt(8-27)-hAmylin(33-37)  28²⁹12Ado-Exendin(1-28)-1des-Lys-hAmylin(1-7)-^(11,18)Arg-sCt(8-27)-hAmylin(33-37) 29²⁹3,6-dioxaoctanoyl-Exendin(1-28)-hAmylin(1-7)-^(11,18)Arg-sCt(8-27)-hAmylin(33-37) 30²⁹3,6-dioxaoctanoyl-Exendin(1-28)-¹des-Lys-hAmylin(1-7),^(11,18)Arg-sCt(8-27)-hAmylin(33-37) 31 ²⁹5Apa-Exendin(1-28)-hAmylin(1-7)-^(11,18)Arg-sCt(8-27)-hAmylin(33-37)  32²⁹5Apa-Exendin(1-28)¹des-Lys-hAmylin(1-7)-^(11,18)Arg-sCt(8-27)-hAmylin(33-37) 33²⁹βAla-βAla-Exendin(1-28),hAmylin(1-7)-^(11,18)Arg-sCt(8-27)-hAmylin(33-37) 34²⁹βAla-βAla-Exendin(1-28)-¹des-Lys-hAmylin(1-7)-^(11,18)Arg-sCt(27)-hAmylin(33-37) 35²⁹4,7,10-trioxa-13-tridecanaminesuccinimidyl-Exendin(1-28)-hAmylin(1-7)^(11,18)Arg-sCt(8-27)-hAmylin(33-37)  36²⁹4,7,10-trioxa-13-tridecanaminesuccinimidyl-Exendin(1-28)-¹des-Lys-hAmylin(1-7)-^(11,18)Arg-sCt(8-27)-hAmylin(33-37)312²⁹(Gly-Gly-Gly)-Exendin(1-28),hAmylin(1-7)-^(11,18)Arg-sCt(8-27)-hAmylin(33-37)313²⁹(Gly-Gly-Gly)-Exendin(1-28)-¹des-Lys-hAmylin(1-7)-^(11,18)Arg-sCt(27)-hAmylin(33-37)

Example 2 Binding Assays

The hybrid polypeptides of the invention may be tested in a variety ofreceptor binding assays using binding assay methodologies generallyknown to those skilled in the art. Such assays include those describedherein.

Amylin binding assay: Evaluation of the binding of some exemplarycompounds of the invention to amylin receptors may be carried out asfollows in nuclueus accumbens membranes prepared from rat brain. MaleSprague-Dawley® rats (200-250) grams are sacrificed by decapitation.Brains are removed and place in cold phosphate-buffered saline (PBS).From the ventral surface, cuts are made rostral to the hypothalamus,bounded laterally by the olfactory tracts and extending at a 45° anglemedially from these tracts. This basal forebrain tissue, containing thenucleus accumbens and surrounding regions, is weighed and homogenized inice-cold 20 mM HEPES buffer (20 mM HEPES acid, pH adjusted to 7.4 withNaOH at 23° C.). Membranes are washed three times in fresh buffer bycentrifugation for 15 minutes at 48,000×g. The final membrane pellet isresuspended in 20 mM HEPES buffer containing 0.2 mM phenylmethylsulfonylfluoride (PMSF).

To measure ¹²⁵I-amylin binding (see, Beaumont K et al. Can J PhysiolPharmacol. 1995 July; 73(7):1025-9), membranes from 4 mg original wetweight of tissue are incubated with ¹²⁵I-amylin at 12-16 pM in 20 mMHEPES buffer containing 0.5 mg/ml bacitracin, 0.5 mg/ml bovine serumalbumin, and 0.2 mM PMSF. Solutions are incubated for 60 minutes at 2°C. Incubations are terminated by filtration through GF/B glass fiberfilters (Whatman Inc., Clifton, N.J.) that are presoaked for 4 hours in0.3% poylethyleneimine in order to reduce nonspecific binding ofradiolabeled peptides. Filters are washed immediately before filtrationwith 5 ml cold PBS, and immediately after filtration with 15 ml coldPBS. Filters are removed and radioactivity assessed in a gamma-counterat a counting efficiency of 77%. Competition curves are generated bymeasuring binding in the presence of 10⁻¹² to 10⁻⁶ M unlabeled testcompound and are analyzed by nonlinear regression using a 4-parameterlogistic equation (Inplot program; GraphPAD Software, San Diego).

CGRP receptor binding assay: Evaluation of the binding of compounds ofthe invention to CGRP receptors are essentially as described for amylinexcept using membranes prepared from SK-N-MC cells, known to expressCGRP receptors (Muff, R. et. al., Ann NY Acad. Sci. 1992: 657, 106-16).Binding assays are performed as described for amylin except using 13,500cpm 125I-hCGRP/well or 21.7 pM/well (Amersham).

Adrenomedullin binding assay: Binding to the adrenomedullin receptor maybe investigated using HUVECs that contain the adrenomedullin receptor(Kato J et. al., Eur J Pharmacol. 1995, 289:383-5) using the PerkinElmer AlphaScreen™ assay for cyclic AMP using an optimum of 25-30,000cells per well. Elevation of cAMP levels is not large for HUVEC comparedto CHO cells. As such, CHO cells may be chosen as a negative controlsince they do not express the adrenomedullin receptor if desired.

Calcitonin receptor binding assay: Binding to the calcitonin receptormay be investigated using CHO cells or T47D cells, which also expressthe calcitonin receptor (Muff R. et. al, Ann N Y Acad Sci. 1992,657:106-16 and Kuestner R. E. et. al. Mol Pharmacol. 1994, 46:246-55),as known in the art.

Leptin binding assay: Two in vitro bioassays are routinely used toassess leptin binding and receptor activation (see e.g., White, et al.,1997. Proc. Natl. Acad. Sci. U.S.A. 94: 10657-10662). An alkalinephosphatase (“AP”)-leptin (“OB”) fusion protein (“AP-OB”) may be used tomeasure inhibition of leptin binding in the absence or presence ofrecombinant mouse leptin (positive control) or peptide, by COS-7 cellstransfected with the long (signaling) form of the mouse OB receptor(“OB-RL”). Signal transduction assays may be done in GT1-7 cellscotransfected with AP reporter and OB-RL constructs. Secreted alkalinephosphatase (“SEAP”) activity in response to stimulation with mouseleptin or peptide may be measured by chemiluminescence.

Y1 receptor binding assay: Membranes are prepared from confluentcultures of SK-N-MC cells that endogenously expresses the neuropeptideY1 receptors. Membranes are incubated with 60 pM [¹²⁵I]-human Peptide YY(2200 Ci/mmol, PerkinElmer Life Sciences), and with unlabeled activecompound for 60 minutes at ambient temperature in a 96 well polystyreneplate. Then well contents are harvested onto a 96 well glass fiber plateusing a Perkin Elmer plate harvestor. Dried glass fiber plates arecombined with scintillant and counted on a Perkin Elmer scintillationcounter.

Y2 receptor binding assay: Membranes are prepared from confluentcultures of SK-N-BE cells that endogenously expresses the neuropeptideY2 receptors. Membranes are incubated with 30 pM [¹²⁵I]-human Peptide YY(2200 Ci/mmol, PerkinElmer Life Sciences), and with unlabeled activecompound for 60 minutes at ambient temperature in a 96 well polystyreneplate. Then well contents are harvested onto a 96 well glass fiber plateusing a Perkin Elmer plate harvestor. Dried glass fiber plates arecombined with scintillant and counted on a Perkin Elmer scintillationcounter.

Y4 receptor binding assay: CHO-Kl cells are transiently transfected withcDNA encoding neuropeptide Y4 gene, and then forty-eight hours latermembranes are prepared from confluent cell cultures. Membranes areincubated with 18 pM [¹²⁵I]-human Pancreatic Polypeptide (2200 Ci/mmol,PerkinElmer Life Sciences), and with unlabeled active compound for 60minutes at ambient temperature in a 96 well polystyrene plate. Then wellcontents are harvested onto a 96 well glass fiber plate using a PerkinElmer plate harvestor. Dried glass fiber plates are combined withscintillant and counted on a Perkin Elmer scintillation counter.

Y5 receptor binding assay: CHO-Kl cells are transiently transfected withcDNA encoding neuropeptide Y5 gene, and then forty-eight hours latermembranes are prepared from confluent cell cultures. Membranes areincubated with 44 pM [¹²⁵I]-human Peptide YY (2200 Ci/mmol, PerkinElmerLife Sciences), and with active compound for 60 minutes at ambienttemperature in a 96 well polystyrene plate. Then well contents areharvested onto a 96 well glass fiber plate using a Perkin Elmer plateharvestor. Dried glass fiber plates are combined with scintillant andcounted on a Perkin Elmer scintillation counter.

GLP-1 receptor binding assay: GLP-1 receptor binding activity andaffinity may be measured using a binding displacement assay in which thereceptor source is RINm5F cell membranes, and the ligand is [¹²⁵I]GLP-1.Homogenized RINm5F cell membranes are incubated in 20 mM HEPES bufferwith 40,000 cpm [¹²⁵I]GLP-1 tracer, and varying concentrations of testcompound for 2 hours at 23° C. with constant mixing. Reaction mixturesare filtered through glass filter pads presoaked with 0.3% PEI solutionand rinsed with ice-cold phosphate buffered saline. Bound counts aredetermined using a scintillation counter. Binding affinities arecalculated using GraphPad Prism software (GraphPad Software, Inc., SanDiego, Calif.).

Example 3 Mouse Food Intake Assay

The hybrid polypeptides of the invention may be tested for appetitesuppression in the mouse food intake assay and for their effect on bodyweight gain in diet-induced obesity (DIO) mice. The experimentalprotocols for the screens are described below.

Female NIH/Swiss mice (8-24 weeks old) are group housed with a 12:12hour light:dark cycle with lights on at 0600. Water and a standardpelleted mouse chow diet are available ad libitum, except as noted.Animals are fasted starting at approximately 1500 hrs, 1 day prior toexperiment. The morning of the experiment, animals are divided intoexperimental groups. In a typical study, n=4 cages with 3 mice/cage.

At time=0 min, all animals are given an intraperitoneal injection ofvehicle or compound, typically in an amount ranging from about 10nmol/kg to 75 nmol/kg, and immediately given a pre-weighed amount (10-15g) of the standard chow. Food is removed and weighed at various times,typically 30, 60, and 120 minutes, to determine the amount of foodconsumed (Morley, Flood et al., Am. J. Physiol. 267: R178-R184, 1994).Food intake is calculated by subtracting the weight of the foodremaining at the e.g., 30, 60, 120, 180 and/or 240 minute time point,from the weight of the food provided initially at time=0. Significanttreatment effects are identified by ANOVA (p<0.05). Where a significantdifference exists, test means are compared to the control mean usingDunnett's test (Prism v. 2.01, GraphPad Software Inc., San Diego,Calif.).

Activity in the food intake assay and sequence of parent molecules usedfor the synthesis of hybrids herein are:

60 min Mouse Food Intake, % ED50 basal Description Cmpd# (nmol/kg)Sequence 30 min 60 min 120 min 180 min Dose PYY(3-36) 1 3IKPEAPGEDASPEELN −31 −38 −40 −26 10 RYYASLRHYLNLVTR nmol/QRY-NH2 (SEQ ID Kg NO: 58) Exendin-4 5 HGEGTFTSDLSKQME −41 −60 −61 −604.8 EEAVRLFIEWLKNGG nmol/ PSSGAPPPS-NH2 (SEQ Kg ID NO: 66)Exendin-4 (1-28) 11 0.3 HGEGTFTSDLSKQME −50 −62 −49 −49 16.3EEAVRLFIEWLKN- nmol/ NH2 (SEQ ID NO: 237) Kg Exendin-4 (1-28) 12 13HGEGAFTSDLSKQLE −53 −61 −50 −53 16.7 [Ala5, Leu14, EEAVRLFIEFLKN- nmol/Phe25] NH2 (SEQ ID NO: 240) Kg Rat Amylin 9 KCNTATCATQRLANF −58 −40−36.5 −35.5 25 LVRSSNNLGPVLPPTN nmol/ VGSNTY-NH2 (SEQ ID Kg NO: 44)hAmylin(1-7)- 10 26 KCNTATCVLGRLSQE −60 −47 −42.5 −32 25^(11,18)Arg-sCT(8- LHRLQTYPRTNTGSN nmol/ 27)-Amylin(33- TY-NH2 (SEQ IDKg 37) NO: 396) CCK-8 26 DY(SO3) MGWMDF- −92 −56 −27 10NH2 (SEQ ID NO: 55) nmol/ Kg

Example 4 Body Weight Gain in Fattened C57Bl/6 (Diet-Induced-Obesity, orDIO) Mice

Male C57BL/6 mice (4 weeks old at start of study) are fed high fat (HF,58% of dietary kcal as fat) or low fat (LF, 11% of dietary kcal as fat)chow. After 4 weeks on chow, each mouse is implanted with an osmoticpump (Alzet #2002) that subcutaneously delivers a predetermined dose ofhybrid polypeptide continuously for two weeks. Body weight and foodintake are measured weekly (Surwit et al., Metabolism—Clinical andExperimental, 44: 645-51, 1995). Effects of the test compound areexpressed as the mean+/−sd of % body weight change (i.e., % change fromstarting weight) of at least 14 mice per treatment group (p<0.05 ANOVA,Dunnett's test, Prism v. 2.01, GraphPad Software Inc., San Diego,Calif.).

Exendin/PYY Hybrids. Exemplary hybrid polypeptides of the invention weresynthesized using a C-terminally truncated exendins (e.g.,exendin-4(1-28) or ⁵Ala,¹⁴Leu,²⁵Phe-exendin-4(1-28)) and an N-terminallytruncated PYY spanning the 18-36 to 31-36 regions. As such, theexemplary hybrid polypeptides generally comprise two modules, whereinthe first module is a fragment of an exendin-4 analog and the secondmodule is a peptidic enhancer selected from PYY truncations. Forcomparison, a β-alanine dipeptide spacers were also incorporated betweenthe peptide building blocks in several variants (see Table 4-1).

TABLE 4-1 Exendin/PYY Hybrids, Receptor Binding Data, and Their Effectsin the Food Intake Assay Receptor Binding (IC50) nM Mouse Food Intake %basal Description Cmpd # Y2 GLP1R 30 min 60 min 120 min Dose PYY(3-36)(SEQ ID NO: 58) 0.04 — −31 −38 −40 −26 ⁵Ala,¹⁴Leu,²⁵Phe-exendin- — 1.9−50 −62 −49 −49 4(1-28) (SEQ ID NO: 240) ⁵Ala,¹⁴Leu,²⁵Phe-exendin- — 1.71000 −4 −11 −10 10 nmol/Kg 4(1-17)-PYY(18-36) ⁵Ala,¹⁴Leu,²⁵Phe-exendin-— 17 2.1 21 9 −4 10 nmol/Kg 4(1-28)-PYY(22-36) (SEQ ID NO: 16)⁵Ala,¹⁴Leu,²⁵Phe-exendin- — 9 0.81 −3 −5 −22 10 nmol/Kg4(1-28)-βAla-βAla- PYY(22-36) (SEQ ID NO: 14) ⁵Ala,¹⁴Leu,²⁵Phe-exendin-2 nd nd 8 −13 −30 10 nmol/Kg 4(1-28)-PYY(25-36) (SEQ ID NO: 15)⁵Ala,¹⁴Leu,²⁵Phe-exendin- 3 13 0.22 −9 −25 −42 10 nmol/Kg4(1-28)-βAla-βAla- PYY(25-36) (SEQ ID NO: 18) ⁵Ala,¹⁴Leu,²⁵Phe-exendin-4 1000 0.25 −14 −36 −52 10 nmol/Kg 4(1-28)-βAla-βAla- PYY(31-36) (SEQ IDNO: 19) exendin-4(1-28)-PYY(25-36) — 16 0.29 −30 −37 −45 10 nmol/Kg (SEQID NO: 8) exendin-4(1-28)-βAla- — 7.8 0.16 −24 −40 −52 10 nmol/KgβAla-PYY(25-36) (SEQ ID NO: 5) exendin-4(1-28)-βAla- — 1000 0.19 −49 −56−61 10 nmol/Kg βAla-PYY(31-36) (SEQ ID NO: 6)

As shown in Table 4-1, certain exemplary compounds of the inventionshowed efficacy in the food intake assay. Certain peptides were alsotested at 75 nmol/kg in the DIO assay and proved to be more efficaciousthan PYY (FIG. 1). As observed for other hybrids herein, hybrids canretain binding to one, two or more receptors that recognize the parentmolecules. Hybrids were designed that recognize at least one receptorfrom each parent or from only one parent, as desired. As observed forother hybrids herein, use of a linker (which can act as a spacer betweeneach adjacent hormone portion) can provide increased activity, includingreceptor(s) binding and in vitro and in vivo activity, such as weightloss. The results herein indicate that a C-terminal portion of PYY canmodulate activity.

Exendin/Amylin Hybrids. Further exemplary hybrid polypeptides of theinvention were prepared from C-terminally truncated exendin (1-27) (SEQID NO:236), C-terminally truncated amylin peptides (e.g., amylin(1-7)(SEQ ID NO:217), ^(2,7)Ala-Amylin(1-7) (SEQ ID NO:285), andAmylin(33-27) (SEQ ID NO:243), and optional sCT fragments (e.g.,sCT(8-10), ^(11,18)Arg-sCT(8-27) (SEQ ID NO:289) and¹⁴Glu,^(11,18)Arg-sCT(8-27) (SEQ ID NO:286). Whereas both hybridpolypeptides were very active in appetite suppression (see Table 4-2),superior to the same dose of rat amylin, the onset of action differedfrom the activity profiles of the parent molecules (data not shown). Ata dose of 1 nmol/kg, Compound 5 was as effective as rat amylin.

TABLE 4-2 Exendin/Amylin Hybrids and Their Effect in the FI AssayReceptor Binding Assay Mouse Food Intake % basal Description Cmpd #GLP-1 Amylin CGRP CT 30 min 60 min 120 min Dose Exendin-4(1- 5 −24 −40−48 25 nmol/Kg 27)-Amylin(1- (SEQ ID 7)-^(11,18)Arg- NO. 25) sCT(8-27)-Amylin(33-37) Exendin-4(1- 6 −40 −59 −66 25 nmol/Kg 27)-^(2,7)Ala- (SEQID Amylin(1-7)- NO. 26) sCT(8-10) Exendin-4(1- 14  0.3 0.2 113 0.1 −5−30 −51 3 nmol/Kg 28)-betaAla- (SEQ ID betaAla- NO: 33) Amylin(1-7)-^(11,18)Arg-sCT(8- 27)-Amylin(33-37) Exendin-4(1- 15  0.4 0.2 63 0.03 −8−36 −51 3 nmol/Kg 28)-Gly-Gly- (SEQ ID Gly-Amylin(1- NO: 312)7)-^(11,18)Arg- sCT(8-27)- Amylin(33-37) Exendin-4(1- 5 1.7 0.6 178 0.5−20 −10 −26 3 nmol/Kg 27)-Amylin(1- (SEQ ID 7)-^(11,18)Arg- NO: 25)sCT(8-27)- Amylin(33-37)

Both compounds also showed excellent efficacy when screened in the DIOassay (FIG. 2).

Further exemplary compounds were assayed for effect on blood glucoselevels and in a food intake assay. These tests included compounds 14 and15. Compound 14, which includes a betaAla linker, is²⁹βAla-βAla-Exendin(1-28),hAmylin(1-7)-^(11,18)Arg-sCt(8-27)-hAmylin(33-37)(SEQ ID NO:33), (alternatively written asExendin(1-28)-βAla-βAla-hAmylin(1-7)-^(11,18)Arg-sCt(8-27)-hAmylin(33-37),while Compound 15 contains a Gly linker:²⁹GlyGlyGly-Exendin(1-28),hAmylin(1-7)-^(11,18)Arg-sCt(8-27)-hAmylin(33-37)(SEQ ID NO:312). The longer exendin(1-28) provided increased activitycompared to exendin(1-27).

A Blood Glucose Assay was performed to test effect on lowering bloodglucose levels. Female NIH/Swiss mice (8-20 weeks old) were group housedwith a 12:12 hour light:dark cycle with lights on at 0600. Water and astandard pelleted mouse chow diet were available ad libitum, except asnoted. The morning of the experiment, animals were divided intoexperimental groups and fasted starting at approximately 0630 hrs. In atypical study, n=2 cages with 3 mice/cage. At time=0 min, a bloodglucose sample was taken and immediately followed by an intraperitonealinjection of vehicle or compound in an amount ranging from about 1nmol/kg to 25 nmol/kg. Blood glucose was measured at 30, 60, 120, 180,and 240 min. Percent pre-treatment was calculated by dividing the bloodglucose at the e.g., 30, 60, 120, 180 and/or 240 minute time point bythe blood glucose at time=0 min. Significant treatment effects wereidentified by ANOVA (p<0.05). Where a significant difference exists,test means were compared to the control mean using Dunnett's test (Prismv. 4.01, GraphPad Software Inc., San Diego, Calif.). The results onexemplary compounds are presented in FIG. 5A. Points represent mean±sd.Peptide was injected intraperitoneally (IP) at t=0 immediately followingbaseline sample into 2-hour fasted NIH/Swiss mice. Samples were taken att=30, 60, 120, 180 and 240 min. Blood glucose was measured with aOneTouch® Ultra® (LifeScan, Inc., a Johnson & Johnson Company, Milpitas,Calif.). * p<0.05 vs. vehicle control; ANOVA, Dunnett's test.

A food intake assay was performed as previously described herein. Theresults are presented in FIG. 5B. Points represent mean±sd of n=4 cages(3 mice/cage). Peptide was injected intraperitoneally (IP) at t=0 intoovernight-fasted NIH/Swiss mice. Food was introduced immediately afterinjection and amount consumed measured at t=30, 60, and 120 min. *p<0.05 vs. vehicle control; ANOVA, Dunnett's test.

Parental Compound 10 and exendin compounds have opposing effects in theGlucose Assay. One would expect that joining them would result incounteracting effects or, at best, a dilution of the effect seen withthe more potent parent compound. However, in the Glucose Assay, theexemplary hybrid compounds were just as efficacious as exendin(1-28) andhad a longer duration of action.

From the food intake data, the exemplary compounds were anorexigenic.Activity was generally better than the parent compounds dosedindividually. Activity was comparable to the parent compounds dosedtogether but at half the concentration of drug (3 nmol/kg hybrid versus6 nmol/kg total for co-dosed parent compounds); thus furtherdemonstrating hybrid superiority. The addition of a linker increasedactivity of the hybrids. The Gly-Gly-Gly linker was more effective thanthe betaAla-betaAla linker in this case.

Exendin/CCK-8 Hybrids. Yet further exemplary hybrid polypeptides of theinvention were prepared from full length or C-terminally truncatedexendin-4 attached to the N-terminus of CCK-8 either directly or via alinker, preserving the N-terminal amide of the CCK-8. (Table 4-3).Further, certain hybrids were prepared incorporating the naturallyoccurring Tyr(SO₃), while another hybrid incorporating the more stablePhe(CH₂SO₃) group was prepared. All the prepared hybrid polypeptideswere active in inhibiting food intake (Table 4-3).

TABLE 4-3 Exendin/CCK-8 Hybrids and Their Effect in the Food IntakeAssay Receptor Binding (IC50) nM Mouse Food Intake % basal DescriptionCmpd # GLP1-R 30 min 60 min 120 min Dose Exendin-4(1-28) 11  0.63 amide(SEQ ID NO: 237) Exendin-4-CCK-8 — −12 −28 −28 10 nmol/Kg (SEQ ID NO.20) Exendin-4(1-28)- 7 75 −20 −36 −45 10 nmol/Kg CCK-8 (SEQ ID NO. 21)Exendin-4(1-28)- 8 31 −24 −47 −66 10 nmol/Kg CCK-8 (SEQ ID [Phe(CH₂SO₃)]NO. 22) Exendin-4(1-28)- 9 −12 −28 −40 10 nmol/Kg [8-amino-3,6- (SEQ IDdioxaoctanoyl]- NO. 23) CCK-8

Exemplary exendin/CCK-8 hybrid polypeptides were tested in the DIO assayat 25 nmol/kg (FIGS. 3A and 3B). The data shows an initial weight loss,followed by a rebound effect in all compounds. Interestingly, therebound effect appears to be diminished in hybrids incorporating themore hydrolytically stable Phe(CH₂SO₃) residue (compare FIGS. 3A and3C), as well as hybrids incorporating a linker, for example the linker8-amino-3,6-dioxaoctanoyl, between the exendin and the CCK residues(compare FIGS. 3A and 3B). A ten-fold greater amount of CCK-8 (250nmol/kg/day) was needed to produce about a −2.8% change at day 2, whichrebounded to the HF diet control level at day 7.

Amylin/PYY Hybrid. An Amylin/PYY hybrid polypeptide was synthesized thatcontained truncated segments of each peptide. In-vivo activity in thefood intake assay is shown in Table 4-4.

TABLE 4-4 Amylin/PYY Phybrid Mouse Food Intake % Basal Description 30min 60 min 120 min Dose Amylin(1-18)-PYY(19-36) −13 −14 −13 25 nmol/Kg(SEQ ID NO: 38)

To ascertain if exemplary hybrid polypeptides of the invention are morepotent than their parent component peptide hormones, exemplary compoundswere tested in the food intake assay at the minimum efficacious dose ofthe more active parent molecule. The results are shown in FIGS. 4A and4B, which also compares the effects of pooled parent peptides (Compounds1, 11, and 12 are component peptide hormones, analogs or fragmentsthereof). The data indicate that several peptides are at least asequipotent as the pooled parent peptides. In parallel with the in vivostudies, in vitro receptor binding and functional assays (cyclaseactivity) have been performed for all the compounds (data not shown).

Amylin-sCT/leptin hybrids. Further exemplary hybrids were made whichcontained a leptin peptide fragment joined to Compound 10, anamylin-sCT-amylin chimera described herein. Compound 16 is [Ser117,dLeu119]leptin(116-122)-[^(11,18)Arg]sCT(8-27)-Amylin(33-37) (SEQ IDNO:397). The compound bound (RBA=receptor binding assay) the amylin andCT receptors, with some binding to the CGRP receptor. The compound wasalso able to activate the CT receptor (C1A assay).

Compound Cmpd# Assay IC50 [Ser117,dLeu119]leptin- 16 amylin RBA  0.04 nM[^(11,18)Arg]sCT(8-27)- amylin(33-37) (SEQ ID NO: 397) 16 CGRP RBA   81nM 16 CT  2.2 nM CYCLASE(C1A) 16 CT RBA (C1A) 0.063 nM 16 GLP RBA (RIN) 1000 nM

This representative molecule was tested for activity in a food intakeassay as described herein. Although leptin was not active in this assay,Compound 16 was anorexigenic at 1 mg/kg. Compound 16 was also superiorto rat amylin (at 25 nmol/kg) in its anorexigenic effect. While Compound10 reduced food intake 91-95% compared to controls, much moreeffectively; Compound 16 reduced the cumulative intake to 34-38% that ofcontrols. Further exemplary embodiments include a head-to-head joiningof the N-terminus of the leptin peptide with that of the[^(11,18)Arg]sCT(8-27)-amylin(33-37) compound.

CCK/Amylin-sCT Hybrids An exemplary hybrid of CCK with an amylin-sCTchimera demonstrated relevant receptor specificity and activation. Theexemplary compound having sequenceDF(P—CH₂SO₃)MGWMDFGKRKCNTATCATQRLANELVRLQTYPRTNVGSNTY (SEQ ID NO:398)demonstrated an IC50 of 0.044 nM in a CT receptor binding assay, 4.4 nMin a CGRP receptor binding assay, 0.083 nM in an amylin receptor bindingassay, and 1000 nM in a GLP Receptor cyclase (RIN).

While the present invention has been described in terms of preferredexamples and embodiments, it is understood that variations andmodifications will occur to those skilled in the art. Therefore, it isintended that the appended claims cover all such equivalent variationswhich come within the scope of the invention as claimed.

1. A hybrid polypeptide exhibiting at least one hormonal activity, saidhybrid polypeptide comprising a first bio-active peptide hormone modulecovalently linked to at least one additional bio-active peptide hormonemodule; wherein: the bio-active peptide hormone modules areindependently selected from the group consisting of: component peptidehormones, fragments of component peptide hormones that exhibit at leastone hormonal activity of the component peptide hormones, analogs andderivatives of component peptide hormones that exhibit at least onehormonal activity of the component peptide hormones, fragments ofanalogs and derivatives of component peptide hormones that exhibit atleast one hormonal activity of the component peptide hormones, andpeptidic enhancers; the component peptide hormones are independentlyselected from at least two of the group consisting of: amylin,adrenomedullin (ADM), calcitonin (CT), calcitonin gene related peptide(CGRP), intermedin, cholecystokinin (CCK), leptin, peptide YY (PYY),glucagon-like peptide-1 (GLP-1), glucagon-like peptide 2 (GLP-2),oxyntomodulin (OXM), a natriuretic peptide, a urocortin family peptide,e.g., Ucn-2 and Ucn-3, a neuromedin family peptide, e.g. neuromedin U25or a splice variant, and ANP, BNP, CNP or urodilatin and exendin-4; thepeptidic enhancers are independently selected from the group consistingof: structural motifs of component peptide hormones that impart adesired chemical stability, conformational stability, metabolicstability, bioavailability, organ/tissue targeting, receptorinteraction, protease inhibition, plasma protein binding, or otherpharmacokinetic characteristic to the hybrid polypeptide, and structuralmotifs of analogs or derivatives of component peptide hormones thatimpart a desired chemical stability, conformational stability, metabolicstability, bioavailability, organ/tissue targeting, receptorinteraction, protease inhibition, plasma protein binding, or otherpharmacokinetic characteristic to the hybrid polypeptide; and at leastone of the bio-active peptide hormone modules exhibits at least onehormonal activity of a component peptide hormone.
 2. The hybridpolypeptide of claim 1, wherein the peptidic enhancers are independentlyselected from the group consisting of: amylin(32-37), amylin(33-37),amylin(34-37), amylin(35-37), amylin(36-37), amylin(37), ADM(47-52),ADM(48-52), ADM(49-52), ADM(50-52), ADM(51-52), ADM(52), CT(27-32),CT(27-32), CT(28-32), CT(29-32), CT(30-32), CT(31-32), CT(32),CGRP(32-37), CGRP(33-37), CGRP(34-37), CGRP(35-37), CGRP(36-37),CGRP(37), intermedin (42-47), intermedin (43-47), intermedin (44-47),intermedin (45-47), intermedin (46-47), intermedin (47), PYY(25-36),PYY(26-36), PYY(27-36), PYY(28-36), PYY(29-36), PYY(30-36), PYY(31-36),PYY(32-36), PYY(25-35), PYY(26-35), PYY(27-35), PYY(28-35), PYY(29-35),PYY(30-35), PYY(31-35), PYY(32-35), frog GLP-1(29-37), frogGLP-1(30-37), frog GLP-2(24-31), exendin-4(31-39), exendin-4(32-39),exendin-4(33-39), exendin-4(34-39), exendin-4(35-39), exendin-4(36-39),exendin-4(37-39), exendin-4(38-39), exendin-4(39), and analogs thereof.3. The hybrid polypeptide of claim 1, wherein at least one of the firstbio-active peptide hormone module or the at least one additionalbio-active peptide hormone module is a component peptide hormone orfragment of a component peptide hormone that exhibits at least onehormonal activity of the component peptide hormone.
 4. The hybridpolypeptide of claim 1, wherein at least one of the first bio-activepeptide hormone module or the at least one additional bio-active peptidehormone module is an analog or derivative of a component peptide hormonethat exhibits at least one hormonal activity or a fragment of an analogor derivative of a component peptide hormone that exhibits at least onehormonal activity of the component peptide hormone.
 5. The hybridpolypeptide of claim 1, wherein at least one of the first bio-activepeptide hormone modules or at least one additional bio-active peptidehormone module is peptidic enhancer.
 6. The hybrid polypeptide of claim1, wherein the component peptide hormones are independently selectedfrom the group consisting of: amylin, calcitonin, CCK, PYY, a urocortinfamily peptide, a neuromedin family peptide, and ANP, BNP, CNP orurodilatin and exendin-4.
 7. The hybrid polypeptide of claim 1, whereinat least one bio-active peptide hormone module that exhibits at leastone hormonal activity is located at the N-terminal portion of the hybridpolypeptide.
 8. The hybrid polypeptide of claim 7, wherein the at leastone bio-active peptide hormone module that exhibits at least onehormonal activity located at the N-terminal portion of the hybridpolypeptide is configured in the C-terminal to N-terminal orientation.9. The hybrid polypeptide of claim 8, wherein the N-terminal portion ofthe hybrid polypeptide is amidated.
 10. The hybrid polypeptide of claim1, wherein at least one bio-active peptide hormone module that exhibitsat least one hormonal activity is located at the C-terminal portion ofthe hybrid polypeptide.
 11. The hybrid polypeptide of claim 10, whereinthe C-terminal end of the hybrid polypeptide is amidated.
 12. The hybridpolypeptide of claim 1, wherein the C-terminal end of one bio-activepeptide hormone module is directly attached to the N-terminal end ofanother bio-active peptide hormone module to form the covalentattachment.
 13. The hybrid polypeptide of claim 1, wherein thebio-active peptide hormone modules are covalently attached using one ormore linking groups independently selected from the group consisting of:alkyls; dicarboxylic acids PEGs; amino acids; polyaminoacids;bifunctional linkers; aminocaproyl (Aca), Gly, β-alanyl,8-amino-3,6-dioxaoctanoyl, and Gly-Lys-Arg (GKR).
 14. The hybridpolypeptide of claim 1, wherein the first bio-active peptide hormonemodule is selected from the group consisting of: exendin-4, a fragmentof exendin-4 that exhibits at least one hormonal activity, an exendin-4analog or derivative that exhibits at least one hormonal activity, and afragment of an exendin-4 analog that exhibits at least one hormonalactivity; and at least one additional bio-active peptide hormone moduleis independently selected from the group consisting of: amylin, afragment of amylin that exhibits at least one hormonal activity, anamylin analog or derivative that exhibits at least one hormonalactivity, or a fragment of an amylin analog that exhibits at least onehormonal activity, CCK, a fragment of CCK that exhibits at least onehormonal activity, a CCK analog or derivative that exhibits at least onehormonal activity, a fragment of a CCK analog that exhibits at least onehormonal activity, CT, a fragment of CT that exhibits at least onehormonal activity, a CT analog or derivative that exhibits at least onehormonal activity, a fragment of a CT analog that exhibits at least onehormonal activity, and a peptidic enhancer.
 15. The hybrid polypeptideof claim 14, wherein the first bio-active peptide hormone module isselected from the group consisting of: exendin-4, exendin-4(1-27),exendin-4(1-28), ¹⁴Leu,²⁵Phe-exendin-4(1-28);⁵Ala,¹⁴Leu,²⁵Phe-exendin-4(1-28) and ¹⁴Leu-exendin-4(1-28); and at leastone additional bio-active peptide hormone module is independentlyselected from the group consisting of: ^(25,28,29)Pro-h-amylin,amylin(1-7), ^(2,7)Ala-amylin(1-7), sCT(8-10), sCT(8-27),^(11,18)Arg-sCT(8-27), ¹⁴Glu,^(11,18)Arg-sCT(8-27), ANP, BNP, CNPurodilatin, CCK-8, Phe²CCK-8, amylin(33-37), PYY(25-36), PYY(30-36) andPYY(31-36).
 16. The hybrid polypeptide of claim 14, wherein the hybridpolypeptide comprises at least three bio-active peptide hormone modules.17. They hybrid polypeptide of claim 14, wherein the hybrid polypeptidecomprises at least four bio-active peptide hormone modules.
 18. Thehybrid polypeptide of claim 14, wherein the first bio-active peptidehormone module is located at the C-terminal end of the hybridpolypeptide and at least one additional bio-active peptide hormonemodule is located at the N-terminal end of the hybrid polypeptide. 19.The hybrid polypeptide of claim 14, wherein the first bio-active peptidehormone module is located at the N-terminal end of the hybridpolypeptide and at least one additional bio-active peptide hormonemodule is located at the C-terminal end of the hybrid polypeptide. 20.The hybrid polypeptide of claim 1, wherein the first bio-active peptidehormone module is selected from the group consisting of: amylin, afragment of amylin that exhibits at least one hormonal activity, anamylin analog or derivative that exhibits at least one hormonalactivity, and a fragment of an amylin analog that exhibits at least onehormonal activity; and at least one additional bio-active peptidehormone module is a peptidic enhancer independently selected from thegroup consisting of: PYY(25-36), PYY(26-36), PYY(27-36), PYY(28-36),PYY(29-36), PYY(30-36), PYY(31-36), PYY(32-36), PYY(25-35), PYY(26-35),PYY(27-35), PYY(28-35), PYY(29-35), PYY(30-35), PYY(31-35), PYY(32-35),and analogs thereof. 21.-25. (canceled)